Anti-angpt2 antibodies

ABSTRACT

The present invention relates to new anti-angiopoietin 2 (ANGPT2) neutralizing antibodies for therapeutic and diagnostic methods and composition using them.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 11, 2019, isnamed 09-0697-US-1_SL.txt and is 78,465 bytes in size.

FIELD OF THE INVENTION

This invention generally relates to anti-angiopoietin 2 (ANGPT2)neutralizing antibodies for diagnostic and therapeutic use. Morespecifically, anti-ANGPT2 antibodies and methods of use for thetreatment of various diseases or disorders characterized by cellsexpressing ANGPT2 are disclosed. Pharmaceutical compositions and kitscomprising the anti-ANGPT2 antibodies are also disclosed.

BACKGROUND OF THE INVENTION

Endothelial dysfunction is a hallmark of chronic kidney disease (CKD)and associated cardiovascular complications. Basic and clinical researchsuggest that improving renal vascular function in CKD will reduceproteinuria and slow the decline in renal function on-top-of SOC. Inaddition, a reduction in ANGPT2 would be expected to elicit a positiveimpact on cardiovascular diseases in CKD patients including in heartfailure, MI, stroke, and others (Eleuteri 2011, Lukasz 2013, Poss 2015,Lorbeer 2015, Tsai 2016, Gerstein 2015, Chen 2009, Chen 2010a, Chen2010b, Gurnik 2016).

The human ANGPT-Tie axis consists of two type-I tyrosine kinasereceptors (Tie1, Tie2) and two secreted ligands (ANGPT1, ANGPT2). ANGPT1is a Tie2 receptor agonist that induces receptor phosphorylation andactivates downstream signaling pathways necessary to preserve renalvascular function, whereas ANGPT2 is a functional antagonist of ANGPT1binding to Tie2. ANGPT1-bound Tie2 is translocated to inter-endothelialcell junctions, where multimeric ANGPT1 can form cross-endothelialcomplexes with Tie2 receptors from adjacent cells to stabilize theglomerular capillary structure. Intracellularly, ANGPT1-induced Tie2phosphorylation leads to the recruitment of adaptor proteins resultingin the activation of AKT survival-promoting pathways and suppressesactivation of the apoptotic pathway.

A small study investigating a role of ANGPT2 in diabetic nephropathy(DN) describe a SNP (1233 A/G) linked to a 20% elevation in circulatingANGPT2 and subsequent increase in DN severity (Quan, 2012). While noANGPT2 blocking antibody has been tested in CKD patients, ANGPT2 mRNAexpression in Pima Indians with CKD was found to be positivelyassociated with interstitial fibrosis and intimal fibrosis. Others haveshown that glomerular ANGPT2 mRNA expression is elevated in diabeticnephropathy (DN) patients as compared to levels from non-diseasedkidneys (Dessapt-Baradez, 2014). However, both ANGPT1 glomerular mRNAexpression (Dessapt-Baradez, 2014) and circulating protein (Chang, 2013;Chang, 2014) are unchanged in DN thereby favoring ANGPT2-bound Tie2 inthe context of disease progression.

Key clinical observations linking dysregulation of the ANGPT2-Tie2pathway with CKD were borne from studies where patients were stratifiedby stage (from stage 1 to ESRD/HD) and where circulating ANGPT2 wasprogressively elevated, correlated with arterial stiffness (Chang,2014), and inversely correlated with a decline in inulin-measuredglomular filtration rate (GFR) (David, 2010). Within a CKD 3-5 patientcohort, ANGPT2 levels were also associated with the severity ofalbuminuria and markers of systemic micro-inflammation (Chang, 2013). Atleast some of the elevated ANGPT2 present in CKD may be due to areduction in miR-145 that normally suppresses ANGPT2 transcription, andwas demonstrated to be significantly reduced in stage 3-5 CKD patients(Chen, 2013). A recent poster presented at American Society ofNephrology (Peters, 2018) showed in non-proliferative diabeticretinopathy patients with baseline albuminuria >30 mg/g that stimulationof Tie2 signaling using the vascular endothelial (VE)—protein tyrosinephosphatase, AKB-9778 (daily s.c. injection), was sufficient to reduceurine albumin-to-creatine ratio (UACR) by approximately 20% in a 3-monthPhase 2A study. These results support that stimulation of the Tie2signaling in severely albuminuric patients is sufficient to reduce CKDprogression. Clinically important values for categorizing CKD patientsare: eGFR of 15-60 ml/min/1.73 m² and UACR of 30 mg/g or greater.

Consistent with the concept that dysregulation of the ANGPT2-Tie2 axiscontributes to CKD, pre-clinical studies demonstrate that geneticmanipulation of either side of the pathway (decreased ANGPT1 orincreased ANGPT2) is sufficient to elicit manifestations of the disease.In mice, conditional deletion of ANGPT1 elicits proteinuric nephropathycharacterized by impaired function of the glomerular filtration barrier,albuminuria, and pathological features seen in humans with advanceddiabetic nephropathy (mesangial matrix expansion and glomerulosclerosis;Jeansson, 2013). Additionally, podocyte-specific ANGPT2 overexpressionresults in increased albuminuria, glomerular endothelial apoptosis, anda reduction in filtration barrier proteins (Davis, 2007). Others haveshown that plasma and renal expression of ANGPT2 are elevated after ⅚nephrectomy in CD1 mice concomitant with elevated ANGPT2 staining inglomeruli (Chang, 2014). The same group demonstrated that the ANGPT2peptibody, L1-10, after ⅚ nephrectomy blocked the vascular expressionand upregulation of multiple pro-fibrotic and pro-inflammatory markersincluding TGFβ1, collagen subtypes, and adhesion molecules althougheffects on renal fibrosis were not interrogated.

ANGPT2 is predominantly expressed in tissues undergoing vascularremodeling and is elevated in the circulation in multiple diseasesconditions, including CKD. Endothelial cells (EC) produce and storeANGPT2 in Weibel-Palade bodies, specialized storage granules from whichANGPT2 can be rapidly released into the circulation to bind blood andlymphatic EC Tie2 receptors (Fiedler, 2004).

Within the normal human kidney, ANGPT2 and Tie2 are expressed on ECsincluding those that face the glomerular basement membrane, withincapillary loops, and ECs within glomeruli. Tie2 is also expressed on ECsadjacent to the podocyte foot process (Satchell, 2002).

The Tie2 agonist ligand, ANGPT1, is secreted from pericytes (Satchell,2001) which surround and support underlying endothelial cells, andimportantly also from podocytes (Satchell, 2002), specialized renalcells that comprise the glomerular filtration barrier thereby enablingcross-talk between podocytes and adjacent glomerular ECs to stabilizethe glomerular capillary structure.

ANGPT2 has limited expression in normal tissues but broad expression inthe actively remodeled vasculature of human tumors. Blocking ANGPT2inhibition of Tie2 signaling is an attractive target for anti-angiogeniccancer therapy and ocular diseases with a vascular basis. Severalantibodies blocking ANGPT2 binding to Tie2 have been developed forclinical use.

Specifically, based on studies with ANGPT2-selective antibodies(REGN910) administered i.v., no dose-limiting safety concerns were notedin Phase I clinical trials (Papadopoulos, 2016). A Tie2-stimulator(AKB-9778) has been tested in Phase II with no noteworthy adverseeffects (AEs) (Campochiaro, 2016), and in multiple clinical studiesthrough Phase III the dual ANGPT1/2 blocker (AMG386) has been testedwith only mild and reversible AEs (Monk, 2014). However, less-selectivetherapeutic approaches with a lower ratio of ANGPT2:ANGPT1 blockade(e.g. AMG386,MEDI3617) were associated with an increased observation ofclinical edema which may be related to dual blockade of lymphatic Tie2receptors as both ANGPT1 and ANGPT2 function as Tie2 receptor agonistsin the lymphatic vasculature. It is believed that blockade of Tie2perturbed the normal flow of the lymphatic and venous circulationleading to extracellular fluid accumulation in general and lymph-edemaspecifically (Monk, 2013). A highly specific ANGPT2 blocking antibodywould be expected to have a significantly diminished risk of edema; noGrade 3-4 edema was observed in Phase I studies with REGN910(Papadopoulos, 2015).

ANGPT2 blockade may impact vascular and lymphatic responsiveness andfunction.

ANGPT2 reportedly plays a role in liver regeneration (Hu, 2014); reducedvascular bed plasticity; altering the healing of liver and other tissues(Gale, 2002) potentially including the tissues of the lung, adipose (An,2017) and ovary (Coxon, 2010); and as an autocrine regulatory switch forendothelial cell inflammatory responses (Fiedler, 2006, Kim, 2016).

The role of ANGPT2 in the adult lymphatic system is not fully known.However, Tie2 is expressed on multiple leukocyte types (monocytes,neutrophils & eosinophils) and modulation of Tie2 signaling may alterimmune sensitivity (Grenga, 2015).

ANGPT2 blockade is an attractive means for preventing other respiratorydisorders including lung vascular hyperpermeability, pulmonary (lung)edema, acute respiratory distress syndrome (ARDS), acute lung injury(ALI), idiopathic interstitial pneumonia, Idiopathic pulmonary fibrosis(IPF) and acute exacerbation IPF, severe acute respiratory syndrome(SARS), and Middle Eastern respiratory syndrome (MERS). High plasmalevel of ANGPT2 plays a central role in the aberrant vascular leakageassociated with plasma increase of Willebrand factor (standard marker ofendothelial injury) in sepsis and ARDS (Calfee, 2012). ANGPT2 andWillebrand Factor plasma levels were significantly elevated in sepsispatients and even higher in ARDS patients (Van der Heijden, 2008).Circulating ANGPT2 was significantly elevated in humans with sepsis whoalso had impaired oxygenation. Serum from these patients disrupted invitro endothelial architecture. This effect of sepsis serum was reversedby ANGPT-1 (ANGPT2 antagonist) (Parikh, 2006). In a mouse model of ARDSinduced by hemorrhagic shock, the pretreatment of the mice with ananti-angpt2 antibody significantly improved lung function, bloodoxygenation and survival rate (Lomas-Neira, 2016)

Increased vascular permeability (vascular hyperpermeability) contributesto many diseases, including ARDS, sepsis, severe sepsis, septic shock,cancer and inflammation. Reducing vascular hyperpermeability of the lungwill reduce the accumulation of fluid in the alveolar space (lung edema)and therefore will improve the gas exchange between the lung and thevessels leading to a better oxygenation of the arterial blood.Improvement of the arterial blood oxygenation translates into a betteroxygenation of all the organs (e.g., brain, heart, liver, kidney) andreduces the risk of multiple organ failure followed by death.

Increase in vascular permeability in sepsis, severe sepsis, septicshock, is also reported in several organs such as lung, kidney, liverand heart. The accumulation of fluid in these organs impairs theirproper functioning (e.g. causing arrhythmia, glomerular filtrationdisruption, or impairment of the metabolism) and leads to organ failurefollowed by death.

Pulmonary (lung) edema is a condition in which the lungs fill withfluid. The most common cause of pulmonary edema is congestive heartfailure. Other less common conditions that may cause pulmonary edemainclude sudden high blood pressure, pneumonia, kidney failure, lungdamage caused by severe infection, severe sepsis of the blood, or bloodpoisoning caused by infection.

Acute lung injury (ALI) is a lung disorder often caused by smokeinhalation including, more recently, in the use of E-cigarette or vapingproducts.

Acute respiratory distress syndrome (ARDS) is a lung inflammationcharacterized by an increase in lung vascular permeability and/or lungedema. ARDS is often characterized as low, mild, or severe based on thedegree of hypoxemia. ARDS can be triggered by several causes, e.g. canbe induced by a bacterial or viral lung infection, by sepsis, inhalationof harmful substances, severe pneumonia, trauma, pancreatitis(inflammation of the pancreas), massive blood transfusions and burns.The most common cause of ARDS is sepsis.

Severe acute respiratory syndrome (SARS) is a viral respiratory illnesscaused by a coronavirus called SARS-associated coronavirus (SARS-CoV).SARS begins with a high fever (temperature greater than 100.4° F.[>38.0° C.]). Other symptoms may include sore throat, cough, headache,an overall feeling of discomfort, and body aches. Some people also havemild respiratory symptoms at the outset. Most patients developpneumonia. Since 2004 until the outbreak of SARS-CoV-2 pandemic inDecember 2019, there have not been any known cases of SARS reportedanywhere in the world.

Middle Eastern respiratory syndrome (MERS) is an illness caused by avirus (more specifically, a coronavirus) called Middle East RespiratorySyndrome Coronavirus (MERS-CoV). The disease is characterized by severerespiratory illness, including fever, cough, and shortness of breath.About three or four out of every 10 patients reported with MERS havedied.

Sepsis, severe sepsis, and septic shock are disorders arising from thesystemic inflammatory response to an infection (see Mitchell M. Levy etal., Crit Care Med. 2003 Apr;31(4):1250-6). Sepsis is a disorder havingboth an infection (e.g., viral, bacterial, abdominal trauma, gutperforation) and a systemic inflammatory response. This leads toincrease in vascular permeability of several organs such as kidneyliver, heart and lung. Severe sepsis (sepsis with organ dysfunction)refers to sepsis with acute organ dysfunction caused by sepsis. Septicshock refers to persistent hypotension unexplained by other causes.

Thus, there is a need for high-affinity neutralizing antibody to ANGPT2that will limit antagonistic binding of ANGPT2 to Tie2, Enhanced Tie2signaling is expected to have many beneficial effects including, forexample, stabilizing the glomerular capillary structure, reducingendothelial activation, and restoring filtration barrier integrity. Intotal, these beneficial effects are expected to decrease proteinuria andpreserve renal function resulting in a slowed disease progression inchronic kidney disease (CKD) patients.

The beneficial effects of a high-affinity neutralizing antibody toANGPT2 are further expected to aid in the treatment of patientsafflicted with vascular hyperpermeability of the lung and associateddisorders.

BRIEF SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies that specificallybind to human ANGPT2. In one aspect of the invention, the antibodies ofthe present invention neutralize ANGPT2. Therefore, the antibodies ofthe invention are useful, for example, for the treatment and/orprevention of diseases or disorders that can be alleviated byneutralizing ANGPT2.

In another aspect, the present invention provides an anti-ANGPT2antibody, in particular a humanized anti-ANGPT2 antibody, having one ormore of the properties described herein below.

In one embodiment, the present invention provides an anti-ANGPT2antibody or an antigen-binding fragment thereof comprising:

a heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 17 (H-CDR3), anda light chain variable region comprising the amino acid sequence of SEQID NO. 19 (L-CDR1); the amino acid sequence of SEQ ID NO. 22 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3),ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 14 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 17 (H-CDR3); anda light chain variable region comprising the amino acid sequence of SEQID NO. 19 (L-CDR1); the amino acid sequence of SEQ ID NO. 22 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3),ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 16 (H-CDR3); anda light chain variable region comprising the amino acid sequence of SEQID NO. 20 (L-CDR1); the amino acid sequence of SEQ ID NO. 23 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3),ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 16 (H-CDR3); anda light chain variable region comprising the amino acid sequence of SEQID NO. 21 (L-CDR1); the amino acid sequence of SEQ ID NO. 23 (L-CDR2);and the amino acid sequence of SEQ ID NO. 25 (L-CDR3),ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 16 (H-CDR3); anda light chain variable region comprising the amino acid sequence of SEQID NO. 20 (L-CDR1); the amino acid sequence of SEQ ID NO. 22 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3).

In another embodiment, the present invention provides an anti-ANGPT2antibody or an antigen-binding fragment thereof comprising:

a variable heavy chain and a variable light chain comprising the aminoacid sequences of SEQ ID NO. 3 and SEQ ID NO. 8, respectively,ora variable heavy chain and a variable light chain comprising the aminoacid sequences of SEQ ID NO. 4 and SEQ ID NO. 9, respectively,ora variable heavy chain and a variable light chain comprising the aminoacid sequences of SEQ ID NO. 5 and SEQ ID NO. 10, respectively, ora variable heavy chain and a variable light chain comprising the aminoacid sequences of SEQ ID NO. 6 and SEQ ID NO. 11, respectively, ora variable heavy chain and a variable light chain comprising the aminoacid sequences of SEQ ID NO. 7 and SEQ ID NO. 12, respectively.

In another embodiment, the present invention provides an anti-ANGPT2antibody or an antigen-binding fragment thereof comprising:

a heavy chain comprising the amino acid sequence of SEQ ID NO. 31 and alight chain comprising the amino acid sequence of SEQ ID NO. 32,ora heavy chain comprising the amino acid sequence of SEQ ID NO. 33 and alight chain comprising the amino acid sequence of SEQ ID NO. 34,ora heavy chain comprising the amino acid sequence of SEQ ID NO. 35 and alight chain comprising the amino acid sequence of SEQ ID NO. 36,ora heavy chain comprising the amino acid sequence of SEQ ID NO. 37 and alight chain comprising the amino acid sequence of SEQ ID NO. 38,ora heavy chain comprising the amino acid sequence of SEQ ID NO. 39 and alight chain comprising the amino acid sequence of SEQ ID NO. 40.

In one embodiment, the present invention provides an anti-ANGPT2antibody or an antigen-binding fragment thereof comprising:

a heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 17 (H-CDR3), ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 14 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 17 (H-CDR3), ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 16 (H-CDR3); ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 16 (H-CDR3); ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 16 (H-CDR3);anda heavy chain framework region comprising one to four amino acidsequences selected from the group consisting of the amino acid sequenceof SEQ ID NO. 41 (H-FR1); amino acid sequence of SEQ ID NO. 42 (H-FR2);the amino acid sequence of SEQ ID NO. 43 (H-FR3); and the amino acidsequence of SEQ ID NO. 44 (H-FR4).

In another embodiment, the present invention provides an anti-ANGPT2antibody or an antigen-binding fragment thereof comprising:

a light chain variable region comprising the amino acid sequence of SEQID NO. 19 (L-CDR1); the amino acid sequence of SEQ ID NO. 22 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3), ora light chain variable region comprising the amino acid sequence of SEQID NO. 19 (L-CDR1); the amino acid sequence of SEQ ID NO. 22 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3), ora light chain variable region comprising the amino acid sequence of SEQID NO. 20 (L-CDR1); the amino acid sequence of SEQ ID NO. 23 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3), ora light chain variable region comprising the amino acid sequence of SEQID NO. 21 (L-CDR1); the amino acid sequence of SEQ ID NO. 23 (L-CDR2);and the amino acid sequence of SEQ ID NO. 25 (L-CDR3), ora light chain variable region comprising the amino acid sequence of SEQID NO. 20 (L-CDR1); the amino acid sequence of SEQ ID NO. 22 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3);anda light chain framework region comprising one to four amino acidsequences selected from the group consisting of the amino acid sequenceof SEQ ID NO. 45 (L-FR1); amino acid sequence of SEQ ID NO. 46 (L-FR2);the amino acid sequence of SEQ ID NO. 47 (L-FR3); and the amino acidsequence of SEQ ID NO. 48 (L-FR4).

In one embodiment, the present invention provides an anti-ANGPT2antibody or antigen-binding fragment thereof that binds to at least oneamino acid residue within amino acid regions 117-148 of thecarboxy-terminal fibrinogen-like domain (FLD) region of human ANGPT2with the SEQ ID NO. 50.

In another embodiment, the invention relates to an ANGTP2 antibody orantigen-binding fragment thereof that binds to SEQ ID NO: 51.

In one embodiment, the anti-ANGPT2 antibody is a humanized anti-ANGPT2antibody.

In another embodiment, the anti-ANGPT2 antibody is a chimericanti-ANGPT2 antibody.

In one embodiment, the present invention provides an anti-ANGPT2antibody or an antigen-binding fragment thereof for use in medicine.

In one embodiment, the present invention provides an anti-ANGPT2antibody or an antigen-binding fragment thereof for use in the treatmentof kidney diseases.

In one embodiment, the present invention provides an anti-ANGPT2antibody or an antigen-binding fragment thereof for use in the treatmentof liver diseases.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising an anti-ANGPT2 antibody or an antigen-bindingfragment thereof and a pharmaceutically acceptable carrier.

In one embodiment, the present invention provides an anti-ANGPT2antibody or an antigen-binding fragment thereof or a pharmaceuticalcomposition comprising the anti-ANGPT2 antibody or an antigen-bindingfragment thereof, wherein said antibody or antigen-binding fragmentthereof is administered by a parenteral route, intravenous route,intravitreal route or subcutaneous route of administration.

In one embodiment, the present invention provides an isolatedpolynucleotide or polynucleotides comprising a sequence encoding a lightchain or light chain variable region of an antibody or antigen-bindingfragment thereof and a heavy chain or heavy chain variable region of anantibody or antigen-binding fragment thereof.

In one embodiment, the present invention provides an expression vectorcomprising an isolated polynucleotide or polynucleotides encoding alight chain or light chain variable region of an antibody orantigen-binding fragment thereof and a heavy chain or heavy chainvariable region of an antibody or antigen-binding fragment thereof.

In one embodiment, the present invention provides a viral vectorcomprising an isolated polynucleotide or polynucleotides encoding alight chain or light chain variable region of an antibody orantigen-binding fragment thereof and a heavy chain or heavy chainvariable region of an antibody or antigen-binding fragment thereof.

In one embodiment, the present invention provides a host cell comprisingan expression vector or an isolated polynucleotide or polynucleotidesencoding a light chain or light chain variable region of an antibody orantigen-binding fragment thereof and a heavy chain or heavy chainvariable region of an antibody or antigen-binding fragment thereof.

In one embodiment, the present invention provides a method for producingan anti-ANGPT2 antibody or an antigen-binding fragment thereofcomprising:

obtaining a host cell comprising an expression vector or an isolatedpolynucleotide or polynucleotides encoding a light chain or light chainvariable region of an antibody or antigen-binding fragment thereof and aheavy chain or heavy chain variable region of an antibody orantigen-binding fragment thereof; and cultivating the host cell.

In one embodiment, the method for producing an anti-ANGPT2 antibody orantigen-binding fragment thereof further comprises recovering andpurifying the anti-ANGPT2 antibody or antigen-binding fragment thereof.

In another embodiment, the present invention relates to an isolatedpolynucleotide or polynucleotides comprising:

a sequence encoding a heavy chain as shown in SEQ ID NO: 31 or a heavychain variable region as shown in SEQ ID NO: 3; and a sequence encodinga light chain as shown in SEQ ID NO. 32 or a light chain variable regionas shown in SEQ ID NO: 8, oran isolated polynucleotide or polynucleotides comprising a sequenceencoding a heavy chain as shown in SEQ ID NO: 33 or a heavy chainvariable region as shown in SEQ ID NO: 4; and a sequence encoding alight chain as shown in SEQ ID NO. 34 or a light chain variable regionas shown in SEQ ID NO: 9,oran isolated polynucleotide or polynucleotides comprising a sequenceencoding a heavy chain as shown in SEQ ID NO: 35 or a heavy chainvariable region as shown in SEQ ID NO: 5; and a sequence encoding alight chain as shown in SEQ ID NO. 36 or a light chain variable regionas shown in SEQ ID NO: 10,oran isolated polynucleotide or polynucleotides comprising a sequenceencoding a heavy chain as shown in SEQ ID NO: 37 or a heavy chainvariable region as shown in SEQ ID NO: 6; and a sequence encoding alight chain as shown in SEQ ID NO. 38 or a light chain variable regionas shown in SEQ ID NO: 11,oran isolated polynucleotide or polynucleotides comprising a sequenceencoding a heavy chain as shown in SEQ ID NO: 39 or a heavy chainvariable region as shown in SEQ ID NO: 7; and a sequence encoding alight chain as shown in SEQ ID NO. 40 or a light chain variable regionas shown in SEQ ID NO: 12.

Non-limiting examples of diseases, disorders, or conditions that can bealleviated by the anti-ANGPT2 antibodies of the invention includecardiac hypertrophy, myocardial infarction, ischemia, ischemicreperfusion injury, stroke hypertension, pulmonary arterialhypertension, idiopathic pulmonary arterial hypertension, trauma inducedbrain disorders, asthma, chronic obstructive pulmonary disease (COPD),rheumatoid arthritis, inflammatory bowel disease, multiplesclerosis,-preeclampsia and pregnancy-induced hypertension, sepsis,severe sepsis, septic shock, non-alcoholic steatohepatitis (NASH),cirrhosis, minimal change disease, focal segmental glomerulosclerosis(FSGS), nephrotic syndrome, diabetic kidney disease (DKD), chronickidney disease (CKD), diabetic renal insufficiency, end stage renaldisease, ischemia or an ischemic reperfusion injury, cancer,hepatocellular carcinoma, idiopathic pulmonary fibrosis (IPF),emphysema, acute lung injury (ALI), acute respiratory disease syndrome(ARDS), severe acute respiratory syndrome (SARS), Middle Easternrespiratory syndrome (MERS), vascular hyperpermeability (and associateddisorders), acute kidney injury (AKI), renal cell carcinoma, heartfailure, lupus nephritis, Raynaud's, pancreatitis, peripheral arterydisease, congenital heart disease, Dengue virus, malaria, hantavirus,edema, regeneration, lupus, interstitial lung disease, scleroderma,retinopathies, diabetic nephropathy, portal hypertension, varicesgrowth, and liver transplantation.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B show the results (duplicate studies) of an angiopoietin2 cell-based complement-dependent cytotoxicity (CDC) assay showing thecytotoxicity of ANGPT2-opt-13 (-□-), an analog of REGN910 (nesvacumab)(-Δ-), an analog of MED13617 (-⋄-), and LC06 (-*-).

FIG. 2 shows the epitope mapping to the FLD domain of anti-ANGPT2 bodies(SEQ ID NO: 59): chimeric lead CL-209881, ANGPT2-opt-13, an analog ofnesvacumab, an analog of MEDI3617, and LC06. Specific binding sites foreach molecule to the extracellular FLD domain of human ANGTP2 (SEQ IDNO: 59) are highlighted in dark grey.

FIGS. 3A-3F shows results for an ANGPT2 blocking assay for ANGPT2-opt-1)(FIG. 3A), ANGPT2-opt-2 (FIG. 3B), ANGPT2-opt-13 (FIG. 3C),ANGPT2-opt-19 (FIG. 3D), ANGPT2-opt-31 (FIG. 3E), chimeric leadCL-209881 (FIG. 3F), and an analog of nesvacumab (FIG. 3G).

DETAILED DESCRIPTION OF THE INVENTION

The generalized structure of antibodies or immunoglobulin is well knownto those of skill in the art, these molecules are heterotetramericglycoproteins, typically of about 150,000 Daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is covalently linked to a heavy chain by one disulfide bondto form a heterodimer, and the heterotrimeric molecule is formed througha covalent disulfide linkage between the two identical heavy chains ofthe heterodimers. Although the light and heavy chains are linkedtogether by one disulfide bond, the number of disulfide linkages betweenthe two heavy chains varies by immunoglobulin isotype. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges. Eachheavy chain has at the amino-terminus a variable domain (V_(H)=variableheavy chain), followed by three or four constant domains (C_(H1),C_(H2), C_(H3), and C_(H4)), as well as a hinge region between C_(H1)and C_(H2). Each light chain has two domains, an amino-terminal variabledomain (V_(L)=variable light chain) and a carboxy-terminal constantdomain (C_(L)). The V_(L) domain associates non-covalently with theV_(H) domain, whereas the C_(L) domain is commonly covalently linked tothe C_(H1) domain via a disulfide bond. Particular amino acid residuesare believed to form an interface between the light and heavy chainvariable domains (Chothia et al., 1985, J. Mol. Biol. 186:651-663.)

Certain domains within the variable domains differ extensively betweendifferent antibodies i.e., are “hypervariable.” These hypervariabledomains contain residues that are directly involved in the binding andspecificity of each particular antibody for its specific antigenicdeterminant. Hypervariability, both in the light chain and the heavychain variable domains, is concentrated in three segments known ascomplementarity determining regions (CDRs) or hypervariable loops(HVLs). CDRs are defined by sequence comparison in Kabat et al., 1991,In: Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., whereasHVLs are structurally defined according to the three-dimensionalstructure of the variable domain, as described by Chothia and Lesk,1987, J. Mol. Biol. 196: 901-917. Where these two methods result inslightly different identifications of a CDR, the structural definitionis preferred. As defined by Kabat, CDR-L1 is positioned at aboutresidues 24-34, CDR-L2, at about residues 50-56, and CDR-L3, at aboutresidues 89-97 in the light chain variable domain; CDR-H1 is positionedat about residues 31-35, CDR-H2 at about residues 50-65, and CDR-H3 atabout residues 95-102 in the heavy chain variable domain. The CDR1,CDR2, CDR3 of the heavy and light chains therefore define the unique andfunctional properties specific for a given antibody.

The three CDRs within each of the heavy and light chains are separatedby framework regions (FR), which contain sequences that tend to be lessvariable. From the amino terminus to the carboxy terminus of the heavyand light chain variable domains, the FRs and CDRs are arranged in theorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The largely β-sheetconfiguration of the FRs brings the CDRs within each of the chains intoclose proximity to each other as well as to the CDRs from the otherchain. The resulting conformation contributes to the antigen bindingsite (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages647-669), although not all CDR residues are necessarily directlyinvolved in antigen binding.

FR residues and Ig constant domains are not directly involved in antigenbinding, but contribute to antigen binding and/or mediate antibodyeffector function. Some FR residues are thought to have a significanteffect on antigen binding in at least three ways: by noncovalentlybinding directly to an epitope, by interacting with one or more CDRresidues, and by affecting the interface between the heavy and lightchains. The constant domains are not directly involved in antigenbinding but mediate various Ig effector functions, such as participationof the antibody in antibody-dependent cellular cytotoxicity (ADCC),complement-dependent cytotoxicity (CDC) and antibody-dependent cellularphagocytosis (ADCP).

The light chains of vertebrate immunoglobulins are assigned to one oftwo clearly distinct classes, kappa (κ) and lambda (λ), based on theamino acid sequence of the constant domain. By comparison, the heavychains of mammalian immunoglobulins are assigned to one of five majorclasses, according to the sequence of the constant domains: IgA, IgD,IgE, IgG, and IgM. IgG and IgA are further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂, respectively.The heavy chain constant domains that correspond to the differentclasses of immunoglobulins are called α, δ, ε, γ, and μ, respectively.The subunit structures and three-dimensional configurations of theclasses of native immunoglobulins are well known.

The terms, “antibody”, “anti-angiopoietin 2 antibody”, “anti-ANGPT2antibody”, “humanized anti-ANGPT2 antibody”, and “variant humanizedanti-ANGPT2 antibody” are used herein in the broadest sense andspecifically encompass monoclonal antibodies (including full lengthmonoclonal antibodies), multispecific antibodies (e.g., bispecificantibodies), antibodies with minor modifications such as N- orC-terminal truncations and antibody fragments such as variable domainsand other portions of antibodies that exhibit a desired biologicalactivity, e.g., ANGPT2 binding.

The term “monoclonal antibody” (mAb) refers to an antibody of apopulation of substantially homogeneous antibodies; that is, theindividual antibodies in that population are identical except fornaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic determinant, an “epitope”. Therefore, the modifier“monoclonal” is indicative of a substantially homogeneous population ofantibodies directed to the identical epitope and is not to be construedas requiring production of the antibody by any particular method. Itshould be understood that monoclonal antibodies can be made by anytechnique or methodology known in the art; including e.g., the hybridomamethod (Kohler et al., 1975, Nature 256:495), or recombinant DNA methodsknown in the art (see, e.g., U.S. Pat. No. 4,816,567), or methods ofisolation of monoclonal recombinantly produced using phage antibodylibraries, using techniques described in Clackson et al., 1991, Nature352: 624-628, and Marks et al., 1991, J. Mol. Biol. 222: 581-597.

Chimeric antibodies consist of the heavy and light chain variableregions of an antibody from one species (e.g., a non-human mammal suchas a mouse) and the heavy and light chain constant regions of anotherspecies (e.g., human) antibody and can be obtained by linking the DNAsequences encoding the variable regions of the antibody from the firstspecies (e.g., mouse) to the DNA sequences for the constant regions ofthe antibody from the second (e.g. human) species and transforming ahost with an expression vector containing the linked sequences to allowit to produce a chimeric antibody. Alternatively, the chimeric antibodyalso could be one in which one or more regions or domains of the heavyand/or light chain is identical with, homologous to, or a variant of thecorresponding sequence in a monoclonal antibody from anotherimmunoglobulin class or isotype, or from a consensus or germlinesequence. Chimeric antibodies can include fragments of such antibodies,provided that the antibody fragment exhibits the desired biologicalactivity of its parent antibody, for example binding to the same epitope(see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc.Natl. Acad. Sci. USA 81: 6851-6855).

The terms, “antibody fragment”, “antigen binding fragment”, “anti-ANGPT2antibody fragment”, “humanized anti-ANGPT2 antibody fragment”, “varianthumanized anti-ANGPT2 antibody fragment” refer to a portion of a fulllength anti-ANGPT2 antibody, in which a variable region or a functionalcapability is retained, for example, specific ANGPT2 epitope binding.Examples of antibody fragments include, but are not limited to, a Fab,Fab′, F(ab′)₂, Fd, Fv, scFv and scFv-Fc fragment, a diabody, a linearantibody, a single-chain antibody, a minibody, a diabody formed fromantibody fragments, and multispecific antibodies formed from antibodyfragments.

Full length antibodies can be treated with enzymes such as papain orpepsin to generate useful antibody fragments. Papain digestion is usedto produce two identical antigen-binding antibody fragments called “Fab”fragments, each with a single antigen-binding site, and a residual “Fc”fragment. The Fab fragment also contains the constant domain of thelight chain and the C_(H1) domain of the heavy chain. Pepsin treatmentyields a F(ab′)₂ fragment that has two antigen-binding sites and isstill capable of cross-linking antigen.

Fab′ fragments differ from Fab fragments by the presence of additionalresidues including one or more cysteines from the antibody hinge regionat the C-terminus of the C_(H1) domain. F(ab′)₂ antibody fragments arepairs of Fab′ fragments linked by cysteine residues in the hinge region.Other chemical couplings of antibody fragments are also known.

“Fv” fragment contains a complete antigen-recognition and binding siteconsisting of a dimer of one heavy and one light chain variable domainin tight, non-covalent association. In this configuration, the threeCDRs of each variable domain interact to define an antigen-biding siteon the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody.

A “single-chain Fv” or “scFv” antibody fragment is a single chain Fvvariant comprising the V_(H) and V_(L) domains of an antibody where thedomains are present in a single polypeptide chain. The single chain Fvis capable of recognizing and binding antigen. The scFv polypeptide mayoptionally also contain a polypeptide linker positioned between theV_(H) and V_(L) domains in order to facilitate formation of a desiredthree-dimensional structure for antigen binding by the scFv (see, e.g.,Pluckthun, 1994, In The Pharmacology of monoclonal Antibodies, Vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315).

Other recognized antibody fragments include those that comprise a pairof tandem Fd segments (V_(H)-C_(H1)-V_(H)-C_(H1)) to form a pair ofantigen binding regions. These “linear antibodies” can be bispecific ormonospecific as described in, for example, Zapata et al. 1995, ProteinEng. 8(10):1057-1062.

A humanized antibody or a humanized antibody fragment is a specific typeof chimeric antibody which includes an immunoglobulin amino acidsequence variant, or fragment thereof, which is capable of binding to apredetermined antigen and which, comprises one or more FRs havingsubstantially the amino acid sequence of a human immunoglobulin and oneor more CDRs having substantially the amino acid sequence of a non-humanimmunoglobulin. This non-human amino acid sequence often referred to asan “import” sequence is typically taken from an “import” antibodydomain, particularly a variable domain. In general, a humanized antibodyincludes at least the CDRs or HVLs of a non-human antibody, insertedbetween the FRs of a human heavy or light chain variable domain.

The present invention describes specific humanized anti-ANGPT2antibodies which contain CDRs derived from the chimeric lead CL-209881inserted between the FRs of human germline sequence heavy and lightchain variable domains.

In one aspect, a humanized anti-ANGPT2 antibody comprises substantiallyall of at least one, and typically two, variable domains (such ascontained, for example, in Fab, Fab′, F(ab′)2, Fabc, and Fv fragments)in which all, or substantially all, of the CDRs correspond to those of anon-human immunoglobulin, and specifically herein, the CDRs are murinesequences of the chimeric lead CL-209881, and the FRs are those of ahuman immunoglobulin consensus or germline sequence. In another aspect,a humanized anti-ANGPT2 antibody also includes at least a portion of animmunoglobulin Fc region, typically that of a human immunoglobulin.Ordinarily, the antibody will contain both the light chain as well as atleast the variable domain of a heavy chain. The antibody also mayinclude one or more of the C_(H1), hinge, C_(H2), C_(H3), and/or C_(H4)regions of the heavy chain, as appropriate.

A humanized anti-ANGPT2 antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂. For example, theconstant domain can be a complement fixing constant domain where it isdesired that the humanized antibody exhibit cytotoxic activity, and theisotype is typically IgG₁. Where such cytotoxic activity is notdesirable, the constant domain may be of another isotype, e.g., IgG₂. Analternative humanized anti-ANGPT2 antibody can comprise sequences frommore than one immunoglobulin class or isotype, and selecting particularconstant domains to optimize desired effector functions is within theordinary skill in the art.

The FRs and CDRs, or HVLs, of a humanized anti-ANGPT2 antibody need notcorrespond precisely to the parental sequences. For example, one or moreresidues in the import CDR, or HVL, or the consensus or germline FRsequence may be altered (e.g., mutagenized) by substitution, insertionor deletion such that the resulting amino acid residue is no longeridentical to the original residue in the corresponding position ineither parental sequence but the antibody nevertheless retains thefunction of binding to ANGPT2. Such alteration typically will not beextensive and will be conservative alterations. Usually, at least 75% ofthe humanized antibody residues will correspond to those of the parentalconsensus or germline FR and import CDR sequences, more often at least90%, and most frequently greater than 95%, or greater than 98% orgreater than 99%.

Immunoglobulin residues that affect the interface between heavy andlight chain variable regions (“the V_(L)-V_(H) interface”) are thosethat affect the proximity or orientation of the two chains with respectto one another. Certain residues that may be involved in interchaininteractions include V_(L) residues 34, 36, 38, 44, 46, 87, 89, 91, 96,and 98 and V_(H) residues 35, 37, 39, 45, 47, 91, 93, 95, 100, and 103(utilizing the numbering system set forth in Kabat et al., Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987)). U.S. Pat. No. 6,407,213 also discusses thatresidues such as V_(L) residues 43 and 85, and V_(H) residues 43 and 60also may be involved in this interaction. While these residues areindicated for human IgG only, they are applicable across species.Important antibody residues that are reasonably expected to be involvedin interchain interactions are selected for substitution into theconsensus sequence.

The terms “consensus sequence” and “consensus antibody” refer to anamino acid sequence which comprises the most frequently occurring aminoacid residue at each location in all immunoglobulins of any particularclass, isotype, or subunit structure, e.g., a human immunoglobulinvariable domain. The consensus sequence may be based on immunoglobulinsof a particular species or of many species. A “consensus” sequence,structure, or antibody is understood to encompass a consensus humansequence as described in certain embodiments, and to refer to an aminoacid sequence which comprises the most frequently occurring amino acidresidues at each location in all human immunoglobulins of any particularclass, isotype, or subunit structure. Thus, the consensus sequencecontains an amino acid sequence having at each position an amino acidthat is present in one or more known immunoglobulins, but which may notexactly duplicate the entire amino acid sequence of any singleimmunoglobulin. The variable region consensus sequence is not obtainedfrom any naturally produced antibody or immunoglobulin. Kabat et al.,1991, Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., andvariants thereof. The FRs of heavy and light chain consensus sequences,and variants thereof, provide useful sequences for the preparation ofhumanized anti-ANGPT2 antibodies. See, for example, U.S. Pat. Nos.6,037,454 and 6,054,297.

Human germline sequences are found naturally in human population. Acombination of those germline genes generates antibody diversity.Germline antibody sequences for the light chain of the antibody comefrom conserved human germline kappa or lambda v-genes and j-genes.Similarly, the heavy chain sequences come from germline v-, d- andj-genes (LeFranc, M-P, and LeFranc, G, “The Immunoglobulin Facts Book”Academic Press, 2001).

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of the antibody's natural environment are thosematerials that may interfere with diagnostic or therapeutic uses of theantibody, and can be enzymes, hormones, or other proteinaceous ornonproteinaceous solutes. In one aspect, the antibody will be purifiedto at least greater than 95% isolation by weight of antibody.

An isolated antibody includes an antibody in situ within recombinantcells in which it is produced, since at least one component of theantibody's natural environment will not be present. Ordinarily however,an isolated antibody will be prepared by at least one purification stepin which the recombinant cellular material is removed.

The term “antibody performance” refers to factors/properties thatcontribute to antibody recognition of antigen or the effectiveness of anantibody in vivo. Changes in the amino acid sequence of an antibody canaffect antibody properties such as folding, and can influence physicalfactors such as initial rate of antibody binding to antigen (k_(a)),dissociation constant of the antibody from antigen (k_(d)), affinityconstant of the antibody for the antigen (Kd), conformation of theantibody, protein stability, and half-life of the antibody.

The term “neutralizing antibody” or “blocking antibody” refers to anantibody whose binding to ANGPT2 blocks the interaction between ANGPT2and its receptor (Tie-2) and/or results in inhibition of at least onebiological function of ANGPT2. It will be understood that the inhibitioncaused by an ANGPT2 neutralizing or blocking antibody need not becomplete so long as it is detectable using an appropriate assay.Exemplary assays for detecting ANGPT2 inhibition are described herein ora known in the art.

As used herein, the terms “identical” or “percent identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of nucleotides or amino acid residues that are thesame, when compared and aligned for maximum correspondence. To determinethe percent identity, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In some embodiments, the two sequences that arecompared are the same length after gaps are introduced within thesequences, as appropriate (e.g., excluding additional sequence extendingbeyond the sequences being compared). For example, when variable regionsequences are compared, the leader and/or constant domain sequences arenot considered. For sequence comparisons between two sequences, a“corresponding” CDR refers to a CDR in the same location in bothsequences (e.g., CDR-H1 of each sequence).

The determination of percent identity or percent similarity between twosequences can be accomplished using a mathematical algorithm. Apreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of two sequences is the algorithm of Karlin and Altschul,1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin andAltschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleicacid encoding a protein of interest. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3, to obtainamino acid sequences homologous to protein of interest. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. Additional algorithms for sequenceanalysis are known in the art and include ADVANCE and ADAM as describedin Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTAdescribed in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. If ktup=2, similar regions in thetwo sequences being compared are found by looking at pairs of alignedresidues; if ktup=1, single aligned amino acids are examined. ktup canbe set to 2 or 1 for protein sequences, or from 1 to 6 for DNAsequences. The default if ktup is not specified is 2 for proteins and 6for DNA. Alternatively, protein sequence alignment may be carried outusing the CLUSTAL W algorithm, as described by Higgins et al., 1996,Methods Enzymol. 266:383-402.

A nucleic acid sequence is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,a nucleic acid presequence or secretory leader is operably linked to anucleic acid encoding a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide; a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and, in the case of a secretory leader,contiguous and in reading frame. However, enhancers are optionallycontiguous. Linking can be accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers can be used.

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably and all such designations include the progenythereof. Thus, “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers.

The term “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domesticated and farm animals,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, andthe like. Preferably, the mammal is human.

A “disorder”, as used herein, is any condition that would benefit fromtreatment with a humanized anti-ANGPT2 antibody described herein. Thisincludes chronic and acute disorders or diseases including thosepathological conditions that predispose the mammal to the disorder inquestion.

As used herein, the term “ANGPT2-associated disorder” or“ANGPT2-associated disease” refers to a condition in which modificationor activation of cells expressing ANGPT2 is indicated. AnANGPT2-associated disorder includes diseases and disorders such asage-related macular degeneration, geographic atrophy, diabeticretinopathy, diabetic macular edema, retinitis pigmentosa, inheritedretinal dystrophy, inherited macular dystrophy, myopic degeneration,retinal vein occlusions, retinal artery occlusions, endophthalmitis,uveitis, cystoid macular edema, choroidal neovascular membrane secondaryto any retinal diseases, optic neuropathies, glaucoma, retinaldetachment, toxic retinopathy, radiation retinopathy, and traumaticretinopathy as well as prodromal and mild-to-moderate Alzheimer'sdiseases, delaying disease progression of patients with Alzheimer'sdisease, Huntington's disease, Parkinson's disease, major depressivedisorder, schizophrenia, cognitive impairment associated withschizophrenia, prevention of first-episode psychosis in individuals withattenuated psychosis syndrome, prevention of relapse in patients withschizophrenia, treatment-resistant depression, and metabolic diseaseslike hyperphagia, obesity or metabolic syndrome.

An ANGPT2-associated disorder also includes cardiac hypertrophy,myocardial infarction, ischemia, ischemic reperfusion injury, strokehypertension, pulmonary arterial hypertension, idiopathic pulmonaryarterial hypertension, trauma induced brain disorders, asthma, chronicobstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatorybowel disease, multiple sclerosis,-preeclampsia and pregnancy-inducedhypertension, sepsis, severe sepsis, septic shock, non-alcoholicsteatohepatitis (NASH), cirrhosis, minimal change disease, focalsegmental glomerulosclerosis (FSGS), nephrotic syndrome, diabetic kidneydisease (DKD), chronic kidney disease (CKD), diabetic renalinsufficiency, end stage renal disease, ischemia or an ischemicreperfusion injury, cancer, hepatocellular carcinoma, idiopathicpulmonary fibrosis (IPF), emphysema, acute lung injury (ALI), acuterespiratory disease syndrome (ARDS), severe acute respiratory syndrome(SARS), Middle Eastern respiratory syndrome (MERS), vascularhyperpermeability (and associated disorders), acute kidney injury, renalcell carcinoma, heart failure, lupus nephritis, Raynaud's, pancreatitis,peripheral artery disease, congenital heart disease, Dengue virus,malaria, hantavirus, edema, regeneration, lupus, interstitial lungdisease, scleroderma, retinopathies, diabetic nephropathy, portalhypertension, varices growth, and liver transplantation.

The term “intravitreal injection” has its normal meaning in the art andrefers to introduction of an anti-ANGPT2 antibody or antigen-bindingfragment thereof into the vitreous of a patient.

The term “specifically binds,” or the like, means that an anti-ANGPT2antibody or antigen-binding fragment thereof forms a complex with anantigen that is relatively stable under physiologic conditions. Methodsfor determining whether two molecules specifically bind are describedherein or a known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. In one embodiment,specific binding is characterized by a K_(D) of about 1×10⁻⁸ M or lessaccording to the Affinity Binding method described in the Examplessection herein. In another embodiment, specific binding is characterizedby a K_(D) of about 1×10⁻⁹ M or less according to the Affinity Bindingmethod described in the Examples section herein. An isolated antibodythat specifically binds human Ang-2 may, however, have cross-reactivityto other antigens, such as ANGPT2-2 molecules from other species.Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The term “subcutaneous administration” refers to introduction of ananti-ANGPT2 antibody or antigen-binding fragment thereof under the skinof an animal or human patient, preferable within a pocket between theskin and underlying tissue, by relatively slow, sustained delivery froma drug receptacle. Pinching or drawing the skin up and away fromunderlying tissue may create the pocket.

The term “subcutaneous infusion” refers to introduction of a drug underthe skin of an animal or human patient, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion may be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human patient, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

The term “subcutaneous bolus” refers to drug administration beneath theskin of an animal or human patient, where bolus drug delivery is lessthan approximately 15 minutes; in another aspect, less than 5 minutes,and in still another aspect, less than 60 seconds. In yet even anotheraspect, administration is within a pocket between the skin andunderlying tissue, where the pocket may be created by pinching ordrawing the skin up and away from underlying tissue.

The term “therapeutically effective amount” is used to refer to anamount of an anti-ANGPT2 antibody or antigen-binding fragment thereofthat relieves or ameliorates one or more of the symptoms of the disorderbeing treated. In doing so, it is that amount that has a beneficialpatient outcome. In one aspect, the therapeutically effective amount hasa neuroprotective or neuroregenerative effect. In another aspect, thetherapeutically effective amount refers to a target serum concentrationthat has been shown to be effective in, for example, slowing diseaseprogression. Efficacy can be measured in conventional ways, depending onthe condition to be treated. For example, efficacy can be measured bydetermining the response rates, e.g. restoration of vision or byassessing the time of delay until disease progression.

The terms “treatment” and “therapy” and the like, as used herein, aremeant to include therapeutic as well as prophylactic, or suppressivemeasures for a disease or disorder leading to any clinically desirableor beneficial effect, including but not limited to alleviation or reliefof one or more symptoms, regression, slowing or cessation of progressionof the disease or disorder. Thus, for example, the term treatmentincludes the administration of an anti-ANGPT2 antibody orantigen-binding fragment thereof prior to or following the onset of asymptom of a disease or disorder thereby preventing or removing one ormore signs of the disease or disorder. As another example, the termincludes the administration of an anti-ANGPT2 antibody orantigen-binding fragment thereof after clinical manifestation of thedisease to combat the symptoms of the disease. Further, administrationof an anti-ANGPT2 antibody or antigen-binding fragment thereof afteronset and after clinical symptoms have developed where administrationaffects clinical parameters of the disease or disorder, such as thedegree of tissue injury or the amount or extent of metastasis, whetheror not the treatment leads to amelioration of the disease, comprises“treatment” or “therapy” as used herein. Moreover, as long as thecompositions of the invention either alone or in combination withanother therapeutic agent alleviate or ameliorate at least one symptomof a disorder being treated as compared to that symptom in the absenceof use of the anti-ANGPT2 antibody composition or antigen-bindingfragment thereof, the result should be considered an effective treatmentof the underlying disorder regardless of whether all the symptoms of thedisorder are alleviated or not.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

Antibodies

Described and disclosed herein are anti-ANGPT2 antibodies, in particularhumanized anti-ANGPT2 antibodies as well as compositions and articles ofmanufacture comprising anti-ANGPT2 antibodies of the present invention.Also described are antigen-binding fragments of an anti-ANGPT2 antibody.The anti-ANGPT2 antibodies and antigen-binding fragments thereof can beused in the treatment of a variety of diseases or disorderscharacterized by reduced activity of the ANGPT2 pathway. An anti-ANGPT2antibody and an antigen-binding fragment thereof each include at least aportion that specifically recognizes an ANGPT2 epitope.

In an initial characterization, the anti-ANGPT2 chimeric lead CL-209881was selected based on its superior antibody performance. A library ofvariants was generated by placing the CDRs of the chimeric lead into FRsof the human consensus heavy and light chain variable domains andfurthermore by engineering the FRs with different alterations.

This resulted in 33 sequences which underwent further optimization andliability-fixing to provide 6 final candidates with the enhancedproperties as disclosed herein. The sequences of the antibody of theinvention as shown below:

VH SEQUENCES CL-209881_VH (chimeric lead), variable heavy chain,SEQ ID NO: 1QVQLKQSGAELVKPGSSVKISCRASGYIFIDYFINWVKQRPGQGLEWIGKIGPGSGSSSSNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAREAFDYDGDYYGMAYWGQGTSVTVSSANGPT2-opt-1 (humanized) variable heavy chain, SEQ ID NO: 3QVQLVQSGAEVKKPGSSVKVSCKASGYIFIDYFINWVRQAPGQGLEWMGKIGPGSGSSSSNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREAFDYEGDYYGMAYWGQGTLVTVSSANGPT2-opt-2 (humanized) variable heavy chain, SEQ ID NO: 4QVQLVQSGAEVKKPGSSVKVSCKASGYIFIEYFINWVRQAPGQGLEWMGKIGPGSGSSSSNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREAFDYEGDYYGMAYWGQGTLVTVSSANGPT2-opt-13 (humanized) variable heavy chain, SEQ ID NO: 5QVQLVQSGAEVKKPGSSVKVSCKASGYIFIDYFINWVRQAPGQGLEWMGKIGPGSGSSSSNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREAFDYDGDYYGMAYWGQGTLVTVSSANGPT2-opt-19 (humanized) variable heavy chain, SEQ ID NO: 6QVQLVQSGAEVKKPGSSVKVSCKASGYIFIDYFINWVRQAPGQGLEWMGKIGPGSGSSSSNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREAFDYDGDYYGMAYWGQGTLVTVSSANGPT2-opt-31 (humanized) variable heavy chain, SEQ ID NO: 7QVQLVQSGAEVKKPGSSVKVSCKASGYIFIDYFINWVRQAPGQGLEWMGKIGPGSGSSSSNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREAFDYDGDYYGMAYWGQGTLVTVSSVL SEQUENCES CL-209881_VL (chimeric lead), variable light chain,SEQ ID NO: 2DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNFLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTITSVQAEDLAVYYCQNDHSYPITFGSGTKLEIKANGPT2-opt-1 (humanized) variable light chain, SEQ ID NO: 8EIVMTQSPATLSVSPGERATLSCKSSQSLLASGNQKNFLAWYQQKPGQAPRLLIYGASTRESGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQNDHSYPITFGQGTKLEIKANGPT2-opt-2 (humanized) variable light chain, SEQ ID NO: 9EIVMTQSPATLSVSPGERATLSCKSSQSLLASGNQKNFLAWYQQKPGQAPRLLIYGASTRESGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQNDHSYPITFGQGTKLEIKANGPT2-opt-13 (humanized) variable light chain, SEQ ID NO: 10EIVMTQSPATLSVSPGERATLSCKSSQSLLSSGNQKSFLAWYQQKPGQAPRLLIYGASTRETGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQNDHSYPITFGQGTKLEIKANGPT2-opt-19 (humanized) variable light chain, SEQ ID NO: 11EIVMTQSPATLSVSPGERATLSCRASQSVLSSGNQKSFLAWYQQKPGQAPRLLIYGASTRETGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQDHSYPITFGQGTKLEIKANGPT2-opt-31 (humanized) variable light chain, SEQ ID NO: 12EIVMTQSPATLSVSPGERATLSCKSSQSLLSSGNQKSFLAWYQQKPGQAPRLLIYGASTRESGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQNDHSYPITFGQGTKLEIK

The underlined portions of the sequences described above correspond tothe CDR regions of the variable light and heavy chain regions.

Humanized anti-ANGPT2 antibodies of the present invention are those thathave the light and heavy chain sequences as set forth in the followingtable.

TABLE 1:  FULL LENGTH LC AND HC SEQUENCES OF THE HUMANIZED ANTI-ANGPT2 ANTIBODIES. Antibody Sequence SEQ ID NO: ANGPT2-opt-1QVQLVQSGAEVKKPGSSVKVSCKASGYIFIDYFINWVR 31 (humanized)QAPGQGLEWMGKIGPGSGSSSSNEKFKGRVTITADKS heavyTSTAYMELSSLRSEDTAVYYCAREAFDYEGDYYGMAY chainWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG ANGPT2-opt-2QVQLVQSGAEVKKPGSSVKVSCKASGYIFIEYFINWVR 33 (humanized)QAPGQGLEWMGKIGPGSGSSSSNEKFKGRVTITADKS heavyTSTAYMELSSLRSEDTAVYYCAREAFDYEGDYYGMAY chainWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG ANGPT2-opt-13QVQLVQSGAEVKKPGSSVKVSCKASGYIFIDYFINWVR 35 (humanized)QAPGQGLEWMGKIGPGSGSSSSNEKFKGRVTITADKS heavyTSTAYMELSSLRSEDTAVYYCAREAFDYDGDYYGMAY chainWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG ANGPT2-opt-19QVQLVQSGAEVKKPGSSVKVSCKASGYIFIDYFINWVR 37 (humanized)QAPGQGLEWMGKIGPGSGSSSSNEKFKGRVTITADKS heavyTSTAYMELSSLRSEDTAVYYCAREAFDYDGDYYGMAY chainWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG ANGPT2-opt-31QVQLVQSGAEVKKPGSSVKVSCKASGYIFIDYFINWVR 39 (humanized)QAPGQGLEWMGKIGPGSGSSSSNEKFKGRVTITADKS heavyTSTAYMELSSLRSEDTAVYYCAREAFDYDGDYYGMAY chainWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG ANGPT2-opt-1EIVMTQSPATLSVSPGERATLSCKSSQSLLASGNQKNF 32 (humanized)LAWYQQKPGQAPRLLIYGASTRESGIPARFSGSGSGT lightEFTLTISSLQSEDFAVYYCQNDHSYPITFGQGTKLEIKR chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECANGPT2-opt-2 EIVMTQSPATLSVSPGERATLSCKSSQSLLASGNQKNF 34 (humanized)LAWYQQKPGQAPRLLIYGASTRESGIPARFSGSGSGT lightEFTLTISSLQSEDFAVYYCQNDHSYPITFGQGTKLEIKR chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECANGPT2-opt-13 EIVMTQSPATLSVSPGERATLSCKSSQSLLSSGNQKSF 36 (humanized)LAWYQQKPGQAPRLLIYGASTRETGIPARFSGSGSGT lightEFTLTISSLQSEDFAVYYCQNDHSYPITFGQGTKLEIKR chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECANGPT2-opt-19 EIVMTQSPATLSVSPGERATLSCRASQSVLSSGNQKSF 38 (humanized)LAWYQQKPGQAPRLLIYGASTRETGIPARFSGSGSGT lightEFTLTISSLQSEDFAVYYCQQDHSYPITFGQGTKLEIKR chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECANGPT2-opt-31 EIVMTQSPATLSVSPGERATLSCKSSQSLLSSGNQKSF 40 (humanized)LAWYQQKPGQAPRLLIYGASTRESGIPARFSGSGSGT lightEFTLTISSLQSEDFAVYYCQNDHSYPITFGQGTKLEIKR chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 2 CDRs OF THE HUMANIZED ANTI-ANGPT2ANTIBODIES ACCORDING TO THE CHEMICAL COMPUTING GROUP (CCG), KLABATHA ANDCHOTHIA NUMBERING SYSTEMS. Antibody Sequence SEQ ID NO: H-CDR1 fromGYIFIDYFIN 13 CL-209881 ANGPT2-opt-1 ANGPT2-opt-13 ANGPT2-opt-19ANGPT2-opt-31 H-CDR1 from GYIFIEYFIN 14 ANGPT2-opt-2 H-CDR2 fromKIGPGSGSSSSNEKFKG 15 CL-209881 ANGPT2-opt-1 ANGPT2-opt-2 ANGPT2-opt-13ANGPT2-opt-19 ANGPT2-opt-31 H-CDR3 from EAFDYDGDYYGMAY 16 CL-209881ANGPT2-opt-13 ANGPT2-opt-19 ANGPT2-opt-31 H-CDR3 from EAFDYEGDYYGMAY 17ANGPT2-opt-1 ANGPT2-opt-2 L-CDR1 from KSSQSLLNSGNQKNFLA 18 CL-209881L-CDR1 from KSSQSLLASGNQKNFLA 19 ANGPT2-opt-1 ANGPT2-opt-2 L-CDR1 fromKSSQSLLSSGNQKSFLA 20 ANGPT2-opt-13 ANGPT2-opt-31 L-CDR1 fromRASQSVLSSGNQKSFLA 21 ANGPT2-opt-19 L-CDR2 from GASTRES 22 CL-209881ANGPT2-opt-1 ANGPT2-opt-2 ANGPT2-opt-31 L-CDR2 from GASTRET 23ANGPT2-opt-13 ANGPT2-opt-19 L-CDR3 from QNDHSYPIT 24 CL-209881ANGPT2-opt-1 ANGPT2-opt-2 ANGPT2-opt-13 ANGPT2-opt-31 L-CDR3 fromQQDHSYPIT 25 BI00767086 H-CDR1 for DYFIN 26 SEQ ID NO. 15 (Kabat)H-CDR1 for EYFIN 27 SEQ ID NO. 16 (Kabat) H-CDR1 for GYIFIDY 28SEQ ID NO. 15 (Chothia) H-CDR1 for GYIFIEY 29 SEQ ID NO. 16 (Chothia)H-CDR2 for GPGSGS 30 SEQ ID NO. 17 (Chothia)

Above CDRs as per the Chemical Computing Group (CCG) numbering areunderlined (Almagro et al., Proteins 2011; 79:3050-3066 and Maier et al,Proteins 2014; 82:1599-1610). The Kabat numbering for the sequences isdenoted by the bold text and the Chothia numbering system by theitalicized text.

Humanization and Amino Acid Sequence Variants

Further variant ANGPT2 antibodies and antibody fragments can beengineered based on the set of CDRs depicted in SEQ ID NOs: 13 to 25. Itis to be understood that in the variant ANGPT2 antibodies and antibodyfragments the amino acid sequence of the CDRs remain unchanged but thesurrounding regions, e.g., FR regions can be engineered. Amino acidsequence variants of the anti-ANGPT2 antibody can be prepared byintroducing appropriate nucleotide changes into the anti-ANGPT2 antibodyDNA, or by peptide synthesis. Such variants include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the anti-ANGPT2 antibodies of theexamples herein. Any combination of deletions, insertions, andsubstitutions is made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid changes also may alter post-translational processes of thehumanized or variant anti-ANGPT2 antibody, such as changing the numberor position of glycosylation sites.

In some embodiments, the present invention includes ANGPT2-antibodies orantibody fragments thereof having a variable heavy chain and a variablelight chain, wherein the variable heavy chain amino acid sequence andthe variable light chain amino acid sequence are at least 80%, at least90%, at least 95%, at least 98%, or at least 99% identical to the aminoacid sequences of SEQ ID NOs. 3 or 8; or 4 or 9; or 5 or 10; or 6 or 11;or 7 or 12, respectively.

In some embodiments, the present invention includes anti-ANGPT2antibodies or antibody fragments thereof having a heavy chain and alight chain, wherein the heavy chain amino acid sequence and the lightchain amino acid sequence are at least 80%, at least 90%, at least 95%,at least 98%, or at least 99% identical to the amino acid sequences ofSEQ ID NOs: 31 or 32; or 33 or 34; or 35 or 36; or 37 or 38; or 39 or40, respectively.

Another type of amino acid variant of the antibody involves altering theoriginal glycosylation pattern of the antibody. The term “altering” inthis context means deleting one or more carbohydrate moieties found inthe antibody, and/or adding one or more glycosylation sites that werenot previously present in the antibody.

In some aspects, the present invention includes nucleic acid moleculesthat encode the amino acid sequence variants of the anti-ANGPT2antibodies described herein. Nucleic acid molecules encoding amino acidsequence variants of the anti-ANGPT2 antibody are prepared by a varietyof methods known in the art. These methods include, but are not limitedto, isolation from a natural source (in the case of naturally occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant version ofthe anti-ANGPT2 antibody.

In certain embodiments, the anti-ANGPT2 antibody is an antibodyfragment. There are techniques that have been developed for theproduction of antibody fragments. Fragments can be derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,1992, Journal of Biochemical and Biophysical Methods 24:107-117; andBrennan et al., 1985, Science 229:81). Alternatively, the fragments canbe produced directly in recombinant host cells. For example, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)₂ fragments (see, e.g., Carter et al., 1992,Bio/Technology 10:163-167). By another approach, F(ab′)₂ fragments canbe isolated directly from recombinant host cell culture. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner.

The anti-ANGPT2 antibodies and antigen-binding fragments thereof caninclude modifications.

In certain embodiments, it may be desirable to use an anti-ANGPT2antibody fragment, rather than an intact antibody. It may be desirableto modify the antibody fragment in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment. In onemethod, the appropriate region of the antibody fragment can be altered(e.g., mutated), or the epitope can be incorporated into a peptide tagthat is then fused to the antibody fragment at either end or in themiddle, for example, by DNA or peptide synthesis. See, e.g., WO96/32478.

In other embodiments, the present invention includes covalentmodifications of the anti-ANGPT2 antibodies. Covalent modificationsinclude modification of cysteinyl residues, histidyl residues, lysinyland amino-terminal residues, arginyl residues, tyrosyl residues,carboxyl side groups (aspartyl or glutamyl), glutaminyl and asparaginylresidues, or seryl, or threonyl residues. Another type of covalentmodification involves chemically or enzymatically coupling glycosides tothe antibody. Such modifications may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody can be introduced intothe molecule by reacting targeted amino acid residues of the antibodywith an organic derivatizing agent that is capable of reacting withselected side chains or the amino- or carboxy-terminal residues.

Removal of any carbohydrate moieties present on the antibody can beaccomplished chemically or enzymatically. Chemical deglycosylation isdescribed by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 andby Edge et al., 1981, Anal. Biochem., 118:131. Enzymatic cleavage ofcarbohydrate moieties on antibodies can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,1987, Meth. Enzymol 138:350.

Another type of useful covalent modification comprises linking theantibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in one or more of U.S. Pat. Nos. 4,640,835, 4,496,689,4,301,144, 4,670,417, 4,791,192 and 4,179,337.

Epitope Binding

In another aspect, the invention relates to an antibody that recognizesa specific “ANGPT2 antigen epitope” and “ANGPT2 epitope”. In particular,the antibody of the invention binds to an epitope in the terminalfibrinogen-like domain (FLD) domain of human ANGTP2 with the SEQ ID NO:50.

In one aspect, the invention relates to a ANGTP2 antibody orantigen-binding fragment thereof that binds to at least one amino acidresidue within the amino acid region 117-148 of the FLD domain of humanANGTP2 (which is in the C-terminus of the full length protein of humanANGPT2) as set forth in SEQ ID NO: 50.

In another aspect, the invention relates to an ANGTP2 antibody orantigen-binding fragment thereof that binds to SEQ ID NO: 51.

The sequences SEQ ID NOs: 50 and 51 are depicted in the following table.

TABLE 3 FLD domain of human ANGPT2 and ANGPT2 epitopeof the antibodies of the invention. SEQ ID Name Sequence NO: FLDKEEQISFRDC AEVFKSGHTT NGIYTLTFPN SEQ domain ofSTEEIKAYCD MEAGGGGWTI IQRREDGSVD ID humanFQRTWKEYKV GFGNPSGEYW LGNEFVSQLT NO: ANGTP2NQQRYVLKIH LKDWEGNEAY SLYEHFYLSS 50 EELNYRIHLK GLTGTAGKIS SISQPGNDFSTKDGDNDKCI CKCSQMLTGG WWFDACGPSN LNGMYYPQRQ NTNKFNGIKW YYWKGSGYSLKATTMMIRPA DF ANGTP2 YLSSEELNYR IHLKGLTGTA GKISSISQPG ND SEQ epitope IDNO: 51

As used herein, the terms “ANGPT2 antigen epitope” and “ANGPT2 epitope”refer to a molecule (e.g., a peptide) or a fragment of a moleculecapable of binding to an anti-ANGPT2 antibody or antigen-bindingfragment thereof. These terms further include, for example, an ANGPT2antigenic determinant recognized by any of the antibodies or antibodyfragments of the present invention, which has a light and heavy chainCDR combination selected from light chain CDRs of the SEQ ID Nos. 18 to25 and heavy chain CDRs of the SEQ ID Nos. 13 to 17. In a furtherembodiment, the ANGPT2 antigenic determinant recognized by any of theantibodies or antibody fragments of the present invention, has a lightand heavy chain CDR combination selected from light chain CDRs of theSEQ ID Nos. 19 to 25 and heavy chain CDRs of the SEQ ID Nos. 13 to 17.

ANGPT2 antigen epitopes can be included in proteins, protein fragments,peptides or the like. The epitopes are most commonly proteins, shortoligopeptides, oligopeptide mimics (i.e., organic compounds that mimicantibody binding properties of the ANGPT2 antigen), or combinationsthereof.

It has been found that the antibodies or antibody fragments of thepresent invention bind to a unique epitope in the FLD domain of humanANGPT2. Preferably, an anti-ANGPT2 antibody or antigen-binding fragmentthereof binds to at least one amino acid residue within the amino acidregion of the FLD domain of human ANGPT2 with the SEQ ID NO: 50.

In one embodiment, the present invention provides an anti-ANGPT2antibody or antigen-binding fragment thereof that binds to at least oneamino acid residue within amino acid regions of the FLD domain of humanANGPT2 with the SEQ ID NO: 51.

Thus, in the context of epitope binding, the phrase “binds within aminoacid region X-Y . . . ” means that the anti-ANGPT2 antibody orantigen-binding fragment thereof binds to at least one amino acidresidue within the amino acid region specified in the sequence.

If for example, the anti-ANGPT2 antibody or antigen-binding fragmentthereof binds to at least one amino acid residue within amino acidregion 117 to 148, this has the meaning that the anti-ANGPT2 antibody orantigen-binding fragment thereof binds to at least one amino acidresidue either within amino acid region 117 to 148 of the FLD domain ofhuman ANPT2 with the SEQ ID NO: 50.

In another aspect, an anti-ANGPT2 antibody or antigen-binding fragmentthereof binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,90%, 95%, or 100% of the amino acid residues within amino acid regions117-148 of the FLD domain of human ANGPT2 with the SEQ ID NO: 50.

FIG. 2 shows the results of epitope mapping for chimeric lead CL_209881,ANGPT2-opt-13 (an exemplary anti-ANGTP2 of the invention), a nesvacumabanalog (where the lysine residue at position 219 is replaced witharginine, K219R), a MED13617 analog, and LC06. Specific binding sitesfor each molecule to the extracellular FLD domain of human ANGTP2 arehighlighted in dark grey. Chimeric lead CL_209881 and ANGPT2-opt-13(which was derived from the CL_209881) bind to an epitope that isdistinct from the epitopes which bind the comparator antibodies.

The anti-ANGPT2 antibodies of the invention block the physicalinteraction between ANGPT2 and Tie2 expressing cells, and show completeinhibition of Tie2 phosphorylation mediated by full length ANGPT2oligomer.

The anti-ANGPT2 antibodies of the invention are highly selective. Thereis no binding to human ANGPT1 FLD domain detected at the highest testedconcentration (500 nM). Furthermore, the anti-ANGPT2 antibodies of theinvention did not show non-specific binding to charged or hydrophobicsurfaces when tested up to 1 μM.

Affinity binding data show that the anti-ANGTP2 antibodies of theinvention have a high-affinity and are highly selective for blockingANGPT2. For example, the anti-ANGPT2 antibodies of the invention havehigh binding affinity to human, cyno and rabbit ANGPT2 FLD domains.

Since the anti-ANGPT2 antibodies of the invention do not show bindingaffinity to recombinant mouse ANGPT2 proteins, mouse efficacy studieswere performed with a mouse cross-reactive tool molecule (the nesvacumabanalog) that recognizes the mouse ANGPT2 FLD domain (K_(D): ˜200 μM). Inthose studies using a disease- and mechanistically-relevant pre-clinicalmodel (db/db UNX mice), treatment with the nesvacumab analog for 8-10weeks, and at dose levels suppressing free circulating ANGPT2, resultedin a significant elevation of glomerular Tie2 phosphorylation and areduction in the disease phenotype including significant improvements inrenal structure (decreases in glomerulosclerosis and interstitialfibrosis).

While the anti-ANGPT2 antibodies of the invention could not be tested inmouse for reductions in nephropathy progression, the weak bindingaffinity of the anti-ANGPT2 antibodies of the invention to the ratANGPT2 FLD domain enabled testing the molecule in an acute mechanisticin vivo permeability model in rat known as a Miles assay. The Milesassay takes advantage of VEGF-mediated ANGPT2 release from storagegranules in endothelial cells that results in a rapid vasculardestabilization in vivo, and an effect that can be quantified; the modelwas validated with the nesvacumab analog. In the rat Miles assay, theanti-permeability effect of ANGPT2-opt-13 was observed (49% reduction inpermeability vs. IgG control), confirming the in vivo activity of themolecule to block ANGPT2-mediated vascular destabilization.

The cytoxicities of the anti-ANGPT2 antibodies of the invention areinvestigated using an ANGPT2 cell-based complement-dependentcytotoxicity (CDC) assay. The CDC assay included two ANGPT1/ANGPT2cross-reactive antibodies (MEDI3617 analog and LC06), a comparatorantibody that does not cross-react with ANGPT1 (nesvacumab analog), andan exemplary anti-ANGPT2 antibody of the invention (ANGPT2-opt-13).

The results of the CDC assay (FIGS. 1A and 1B), show that theANGPT1/ANGPT2 cross-reactive comparator antibodies (MEDI3617 analog andLC06) and ANGPT2 specific comparator antibody (nesvacumab analog) allexhibit higher cytotoxicities than does ANGPT2-opt-13.

Therapeutic Uses

In one embodiment, the anti-ANGPT2 antibodies of the invention, orantigen-binding fragments thereof are useful for treating or preventingdiseases or disorders that can be alleviated by neutralizing ANGPT2(“the ANGPT2 related diseases or disorders”).

In another embodiment, the anti-ANGPT2 antibodies of the invention, orantigen-binding fragments thereof, are useful as a medicament.

In one embodiment, the ANGPT2 disease or disorder is selected from thegroup consisting of An ANGPT2-associated disorder also includes cardiachypertrophy, myocardial infarction, ischemia, ischemic reperfusioninjury, stroke hypertension, pulmonary arterial hypertension, idiopathicpulmonary arterial hypertension, trauma induced brain disorders, asthma,chronic obstructive pulmonary disease (COPD), rheumatoid arthritis,inflammatory bowel disease, multiple sclerosis,-preeclampsia andpregnancy-induced hypertension, sepsis, severe sepsis, septic shock,non-alcoholic steatohepatitis (NASH), cirrhosis, minimal change disease,focal segmental glomerulosclerosis (FSGS), nephrotic syndrome, diabetickidney disease (DKD), chronic kidney disease (CKD), diabetic renalinsufficiency, end stage renal disease, ischemia or an ischemicreperfusion injury, cancer, hepatocellular carcinoma, idiopathicpulmonary fibrosis (IPF), emphysema, acute lung injury (ALI), acuterespiratory disease syndrome (ARDS), severe acute respiratory syndrome(SARS), Middle Eastern respiratory syndrome (MERS), vascularhyperpermeability (and associated disorders), acute kidney injury, renalcell carcinoma, heart failure, lupus nephritis, Raynaud's, pancreatitis,peripheral artery disease, congenital heart disease, Dengue virus,malaria, hantavirus, edema, regeneration, lupus, interstitial lungdisease, scleroderma, retinopathies, diabetic nephropathy, portalhypertension, varices growth, and liver transplantation.

In one embodiment, the present invention relates to a method fortreating NASH, cirrhosis, portal hypertension, varices growth varicealhemorrhage, and hepatic encephalopathy.

In another embodiment, the present invention relates to a method fortreating chronic kidney disease. The term “chronic kidney disease”generally refers to a patient having reducing kidney function asmeasured by glomular filtration rate (GFR). An estimated GFR (eGFR) of90 or greater (Stage 1) is considered normal kidney function; an eGFR or89 to 60 (Stage 2) is considered mild loss of kidney function; an eGFRof 59 to 45 (Stage 3a) is considered mild to moderate loss of kidneyfunction; an eGFR of 44 to 30 (Stage 3b) is considered moderate tosevere loss of kidney function; and a GFR of 29 to 15 (Stage 4) isconsidered severe loss of kidney function.

In another embodiment, the CKD patients have an albumin-to-creatineratio (UACR)≥30 mg/g and an eGFR of 20-75 ml/min/1.73 m² or 15-60ml/min/1.73 m².

In one embodiment, the invention relates to the treatment of Stage 2chronic kidney disease; in another embodiment, the invention relates tothe treatment of Stage 3a chronic kidney disease; in another embodiment,the invention relates to the treatment of Stage 3b chronic kidneydisease; and in another embodiment, the invention relates to thetreatment of Stage 4 chronic kidney disease.

In another embodiment, the invention relates to the treatment of chronickidney disease in a patient having an ≥eGFR 60; in another embodiment,the invention relates to the treatment of chronic kidney disease in apatient having an ≥eGFR 45; in another embodiment, the invention relatesto the treatment of chronic kidney disease in a patient having an ≥eGFR30; and in another embodiment, the invention relates to the treatment ofchronic kidney disease in a patient having an ≥eGFR 20.

In another embodiment, the invention relates to the treatment of chronickidney disease in a patient having an eGFR of 20-75 ml/min/1.73 m2.

In another embodiment, the invention relates to a method for treatingchronic kidney disease in a patient having a UACR≥30 mg/g.

In another embodiment, the invention relates to a method for treatingchronic kidney disease in rapid progressing (fast progressor) patients.As used herein, the term rapid progressing CKD patients have an eGFR of20-75 ml/min/1.73 m2 and a decline ≥3 ml/min/1.73 m2/year. In anotherembodiment, the rapid progressing CKD patients have an eGFR of 20-75ml/min/1.73 m2 and a decline ≥4 ml/min/1.73 m2/year.

In another embodiment, the invention relates to a method for reducingprogression to end stage renal disease (ESRD), dialysis and/orcardiovascular events in a CKD patent having an eGFR 20-75 ml/min/1.73m2.

In another embodiment, the invention relates to a method for reducingrisk of a cardiovascular event in a CKD patient, for example, a patienthaving an eGFR 20-75 ml/min/1.73 m2.

In another embodiment, the present invention relates to a method fortreating diabetic nephropathy.

In another embodiment, the present invention relates to methods fortreating vascular hyperpermeability and/or its associated disorders.These include (i) respiratory disorders associated with vascularhyperpermeability that are not primarily caused by an infection (Group1), and (ii) respiratory disorders associated with vascularhyperpermeability caused by certain bacterial, viral, or fungalparasites infections (Group 2).

Nonlimiting respiratory disorders associated with vascularhyperpermeability of Group 1 include pulmonary (lung) edema, idiopathicinterstitial pneumonia, IPF and acute exacerbation IPF, ARDS notinfection-related, and ALI; and

Nonlimiting respiratory disorders associated with vascularhyperpermeability of Group 2 include ARDS related to an infection, SARS,MERS, sepsis, severe sepsis, and septic shock.

ARDS, not infection-related, is understood as ARDS which is nottriggered or caused by an infection, such as ARDS caused by inhalationof harmful substances (e.g. toxic smoke), trauma, pancreatitis, gastricjuice reflux, massive blood transfusions or burns.

ARDS, infection-related, is understood as ARDS which is triggered orcaused by an infection, such as ARDS caused by sepsis or severepneumonia.

In one embodiment, the present invention relates to treatment ofvascular hyperpermeability.

In another embodiment, the present invention relates treatment ofvascular hyperpermeability arising from or caused by a bacterialinfection including, but not limited to, Legionella pneumophila,Haemophilus influenzae, Sterptococcus pneumonia, Klebsiella, Mycoplasmapneumonia, and Staphylococcus aureus.

In another embodiment, the present invention relates treatment ofvascular hyperpermeability arising from or caused by a fungal infectionincluding, but not limited to, fungal pneumonia and parasitic pneumonia.

In another embodiment, the present invention relates treatment ofvascular hyperpermeability arising from or caused by a viral infectionincluding, not limited to, influenza H1N1, respiratory syncytial virus,parainfluenza, adenovirus, and human coronavirus (CoV) infections suchas SARS-CoV, SARS-CoV-2 and MERS-CoV.

In another embodiment, the present invention relates to treatment ofvascular hyperpermeability associated with ALI, ARDS, or SARS.

Non-Therapeutic Uses

The antibodies described herein are useful as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a Protein A resin, using methods well known in the art. Theimmobilized antibody is contacted with a sample containing the ANGPT2protein (or fragment thereof) to be purified, and thereafter the supportis washed with a suitable solvent that will remove substantially all thematerial in the sample except the ANGPT2 protein, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the ANGPT2 protein from the antibody.

Anti-ANGPT2 antibodies are also useful in diagnostic assays to detectand/or quantify ANGPT2 protein, for example, detecting ANGPT2 expressionin specific cells, tissues, or serum.

It will be advantageous in some embodiments, for example, for diagnosticpurposes to label the antibody with a detectable moiety. Numerousdetectable labels are available, including radioisotopes, fluorescentlabels, enzyme substrate labels and the like. The label may beindirectly conjugated with the antibody using various known techniques.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody can be conjugated with a small hapten (such asdigoxin) and one of the different types of labels mentioned above isconjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).Thus, indirect conjugation of the label with the antibody can beachieved.

Exemplary radioisotopes labels include ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. Theantibody can be labeled with the radioisotope, using the techniquesdescribed in, for example, Current Protocols in Immunology, Volumes 1and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N.Y.,Pubs. Radioactivity can be measured, for example, by scintillationcounting.

Exemplary fluorescent labels include labels derived from rare earthchelates (europium chelates) or fluorescein and its derivatives,rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, andTexas Red are available. The fluorescent labels can be conjugated to theantibody via known techniques, such as those disclosed in CurrentProtocols in Immunology, supra, for example. Fluorescence can bequantified using a fluorimeter.

There are various well-characterized enzyme-substrate labels known inthe art (see, e.g., U.S. Pat. No. 4,275,149 for a review). The enzymegenerally catalyzes a chemical alteration of the chromogenic substratethat can be measured using various techniques. For example, alterationmay be a color change in a substrate that can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light that can be measured, using achemiluminometer, for example, or donates energy to a fluorescentacceptor.

Examples of enzymatic labels include luciferases such as fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (such asglucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocydic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described, for example, inO'Sullivan et al., 1981, Methods for the Preparation of Enzyme-AntibodyConjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J.Langone & H. Van Vunakis, eds.), Academic press, N.Y., 73: 147-166.

Examples of enzyme-substrate combinations include, for example:Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor such asorthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB); alkaline phosphatase (AP) with para-Nitrophenylphosphate as chromogenic substrate; and β-D-galactosidase (β-D-Gal) witha chromogenic substrate such as p-nitrophenyl-β-D-galactosidase orfluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

In another embodiment, the anti-ANGPT2 antibody is used unlabeled anddetected with a labeled antibody that binds the anti-ANGPT2 antibody.

The antibodies described herein may be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. See, e.g., Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).Diagnostic Kits.

A humanized anti-ANGPT2 antibody can be used in a diagnostic kit, i.e.,a packaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibody islabeled with an enzyme, the kit may include substrates and cofactorsrequired by the enzyme such as a substrate precursor that provides thedetectable chromophore or fluorophore. In addition, other additives maybe included such as stabilizers, buffers (for example a block buffer orlysis buffer), and the like. The relative amounts of the variousreagents may be varied widely to provide for concentrations in solutionof the reagents that substantially optimize the sensitivity of theassay. The reagents may be provided as dry powders, usually lyophilized,including excipients that on dissolution will provide a reagent solutionhaving the appropriate concentration.

Diagnostic Kits

An anti-ANGPT2 antibody can be used in a diagnostic kit, i.e., apackaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibody islabeled with an enzyme, the kit may include substrates and cofactorsrequired by the enzyme such as a substrate precursor that provides thedetectable chromophore or fluorophore. In addition, other additives maybe included such as stabilizers, buffers (for example a block buffer orlysis buffer), and the like. The relative amounts of the variousreagents may be varied widely to provide for concentrations in solutionof the reagents that substantially optimize the sensitivity of theassay. The reagents may be provided as dry powders, usually lyophilized,including excipients that on dissolution will provide a reagent solutionhaving the appropriate concentration.

Compositions and Administration Thereof

A composition comprising an anti-ANGPT2 antibody or an antigen-bindingfragment thereof can be administered to a subject having or at risk ofthe ANGPT2 related diseases or disorders described herein. The inventionfurther provides for the use of an anti-ANGPT2 antibody or anantigen-binding fragment thereof in the manufacture of a medicament forprevention or treatment of an ANGPT2 disease. The term “subject” as usedherein means any mammalian patient to which an anti-ANGPT2 antibody oran antigen-binding fragment thereof can be administered, including,e.g., humans and certain non-human mammals, such as primates, and dogs.Subjects specifically intended for treatment using the methods describedherein include humans. The anti-ANGPT2 antibody or an antigen-bindingfragment thereof can be administered either alone or in combination withother compositions.

Preferred antibodies for use in such pharmaceutical compositions arethose that comprise the antibody according to the invention.

Various delivery systems are known and can be used to administer theanti-ANGPT2 antibody or an antigen-binding fragment thereof. Methods ofintroduction include but are not limited to intravitreal, eye drops,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, and oral routes. The anti-ANGPT2 antibody or anantigen-binding fragment thereof can be administered, for example byinfusion, bolus or injection, and can be administered together withother biologically active agents. Administration can be systemic orlocal. In preferred embodiments, the administration is by intravitrealinjection. Formulations for such injections may be prepared in, forexample, prefilled syringes.

An anti-ANGPT2 antibody or an antigen-binding fragment thereof can beadministered as pharmaceutical compositions comprising a therapeuticallyeffective amount of the anti-ANGPT2 antibody or an antigen-bindingfragment thereof and one or more pharmaceutically compatibleingredients.

In typical embodiments, the pharmaceutical composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous or subcutaneous administration to human beings.Typically, compositions for administration by injection are solutions insterile isotonic aqueous buffer. Where necessary, the pharmaceutical canalso include a solubilizing agent and a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where thepharmaceutical is to be administered by infusion, it can be dispensedwith an infusion bottle containing sterile pharmaceutical grade water orsaline. Where the pharmaceutical is administered by injection, anampoule of sterile water for injection or saline can be provided so thatthe ingredients can be mixed prior to administration.

Further, the pharmaceutical composition can be provided as apharmaceutical kit comprising (a) a container containing an anti-ANGPT2antibody or an antigen-binding fragment thereof in lyophilized form and(b) a second container containing a pharmaceutically acceptable diluent(e.g., sterile water) for injection. The pharmaceutically acceptablediluent can be used for reconstitution or dilution of the lyophilizedanti-ANGPT2 antibody or antigen-binding fragment thereof. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The amount of the anti-ANGPT2 antibody or antigen-binding fragmentthereof that is effective in the treatment or prevention an ANGPT2related diseases or disorders can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and thestage of disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems.

For example, toxicity and therapeutic efficacy of the anti-ANGPT2antibody or antigen-binding fragment thereof can be determined in cellcultures or experimental animals by standard pharmaceutical proceduresfor determining the ED₅₀ (the dose therapeutically effective in 50% ofthe population). An anti-ANGPT2 antibody or antigen-binding fragmentthereof that exhibits a large therapeutic index is preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofthe anti-ANGPT2 antibody or antigen-binding fragment thereof typicallylies within a range of circulating concentrations that include the ED₅₀with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any anti-ANGPT2 antibody or antigen-binding fragmentthereof used in the method, the therapeutically effective dose can beestimated initially from cell culture assays. A dose can be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC₅₀ (i.e., the concentration of the test compoundthat achieves a half-maximal inhibition of symptoms) as determined incell culture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma can be measured, for example,by high performance liquid chromatography, ELISA and the like.

For intravitreal injection of the ANGPT2-antibody generally longerintervals between treatments are preferred. Due to its improved potencythe ANGPT2 antibodies of the present invention can be administered inlonger intervals.

In one embodiment, the ANGPT2-antibody is administered every 6 weeks, orevery 7 weeks, or every 8 weeks, or every 9 weeks, or every 10 weeks, orevery 11 weeks, or every 12 weeks. In a further embodiment, theANGPT2-antibody of the invention is administered once every 3 months.

Antibodies of the present invention can be formulated to doses whichinclude, but are not limited from 20 mg/ml to 180 mg/ml; or 20 mg/ml, 30mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, or100 mg/ml. Preferably, antibodies of the present invention can beformulated in a liquid formulation of about 50 mg/ml to of about 150mg/ml.

In some embodiments, the pharmaceutical compositions comprising theanti-ANGPT2 antibody or antigen-binding fragment thereof can furthercomprise a therapeutic agent, either conjugated or unconjugated to thebinding agent.

Such combination therapy administration can have an additive orsynergistic effect on disease parameters (e.g., severity of a symptom,the number of symptoms, or frequency of relapse).

With respect to therapeutic regimens for combinatorial administration,in a specific embodiment, an anti-ANGPT2 antibody or antigen-bindingfragment thereof is administered concurrently with a therapeutic agent.In another specific embodiment, the therapeutic agent is administeredprior or subsequent to administration of the anti-ANGPT2 antibody orantigen-binding fragment thereof, by at least an hour and up to severalmonths, for example at least an hour, five hours, 12 hours, a day, aweek, a month, or three months, prior or subsequent to administration ofthe anti-ANGPT2 antibody or antigen-binding fragment thereof.

The compounds of the invention may be used alone or in combination ofone or more additional therapeutic agents. Nonlimiting examples ofadditional therapeutic agents may include:

antidiabetics such as alpha-glucosidase inhibitors (e.g., miglitol andacarbose), amylin analogs (e.g., pramlintide), dipeptidyl peptidase 4inhibitors (e.g., alogliptin, sitagliptin, saxagliptin, andlinagliptin), incretin mimetics (e.g., liraglutide, exenatide,liraglutide, exenatide, dulaglutide, albiglutide, and lixisenatide),insulin, meglitinides (e.g., repaglinide and nateglinide), biguanides(e.g., metformin); SGLT-2 inhibitors (e.g., canagliflozin,empagliflozin, and dapagliflozin), sulfonylureas (e.g., chlorpropamide,glimepiride, glyburide, glipizide, glyburide, tolazamide, andtolbutamide), and thiazolidinediones (e.g., rosiglitazone andpioglitazone);

angiotensin II receptor antagonists (angiotensin receptor blockers(ARBs)) such as candesartan, eprosartan, candesartan, irbesartan,losartan, olmesartan, telmisartan, valsartan, azilsartan, and medoxomil;

angiotensin converting enzyme inhibitors (e.g., benazepril, captopril,enalapril, fosinopril, lisinopril, moexipril, and perindopril);

anticoagulants (e.g. dabigatran, actylise, Warfarin, heparin, andacetylsalicylic acid);

bronchodilators including short-acting and long-action beta agonists(e.g., albuterol, levalbuterol, salmeterol, formoterol, arformoterol,vilanterol, indacaterol and olodaterol) and short- and long-actinganticholinergics (ipratropium, tiotropium, umeclidinium, glycopyrrolateiand aclidinium);

steroids such as fluticasone and budesonide;

antimalarials such as hydroxychloroquine or chloroquine;

virostatic nucleosid analogs such as remdesivir; and

HIV-protease inhibitors such as lopinavir-ritonavir;

Polynucleotides, Vectors, Host Cells, and Recombinant Methods

The present invention relates to isolated polynucleotides that comprisea sequence encoding an anti-ANGPT2 antibody, vectors, and host cellscomprising the polynucleotides, and recombinant techniques forproduction of the antibody. The isolated polynucleotides can encode anydesired form of the anti-ANGPT2 antibody including, for example, fulllength monoclonal antibodies, Fab, Fab′, F(ab′)₂, and Fv fragments,diabodies, linear antibodies, single-chain antibody molecules, andmultispecific antibodies formed from antibody fragments.

The polynucleotide(s) that comprise a sequence encoding an anti-ANGPT2antibody or a fragment or chain thereof can be fused to one or moreregulatory or control sequence, as known in the art, and can becontained in suitable expression vectors or host cell as known in theart. Each of the polynucleotide molecules encoding the heavy or lightchain variable domains can be independently fused to a polynucleotidesequence encoding a constant domain, such as a human constant domain,enabling the production of intact antibodies. Alternatively,polynucleotides, or portions thereof, can be fused together, providing atemplate for production of a single chain antibody.

For recombinant production, a polynucleotide encoding the antibody isinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Many suitable vectors for expressing the recombinantantibody are available. The vector components generally include, but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

The anti-ANGPT2 antibodies can also be produced as fusion polypeptides,in which the antibody is fused with a heterologous polypeptide, such asa signal sequence or other polypeptide having a specific cleavage siteat the amino terminus of the mature protein or polypeptide. Theheterologous signal sequence selected is typically one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe anti-ANGPT2 antibody signal sequence, the signal sequence can besubstituted by a prokaryotic signal sequence. The signal sequence canbe, for example, alkaline phosphatase, penicillinase, lipoprotein,heat-stable enterotoxin II leaders, and the like. For yeast secretion,the native signal sequence can be substituted, for example, with aleader sequence obtained from yeast invertase alpha-factor (includingSaccharomyces and Kluyveromyces α-factor leaders), acid phosphatase, C.albicans glucoamylase, or the signal described in WO90/13646. Inmammalian cells, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, can be used. The DNAfor such precursor region is ligated in reading frame to DNA encodingthe humanized anti-ANGPT2 antibody.

Expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2-ν. plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV, andBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a gene that encodes aselectable marker to facilitate identification of expression. Typicalselectable marker genes encode proteins that confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, or alternatively, are complement auxotrophicdeficiencies, or in other alternatives supply specific nutrients thatare not present in complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid, and hygromycin. Common selectablemarkers for mammalian cells are those that enable the identification ofcells competent to take up a nucleic acid encoding a humanizedanti-ANGPT2 antibody, such as DHFR (dihydrofolate reductase), thymidinekinase, metallothionein-I and -II (such as primate metallothioneingenes), adenosine deaminase, ornithine decarboxylase, and the like.Cells transformed with the DHFR selection gene are first identified byculturing all of the transformants in a culture medium that containsmethotrexate (Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity (e.g., DG44).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding anti- ANGPT2 antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH),can be selected by cell growth in medium containing a selection agentfor the selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See, e.g., U.S. Pat. No. 4,965,199.

Where the recombinant production is performed in a yeast cell as a hostcell, the TRP1 gene present in the yeast plasmid YRp7 (Stinchcomb etal., 1979, Nature 282: 39) can be used as a selectable marker. The TRP1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1(Jones, 1977, Genetics 85:12). The presence of the trp1 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626are complemented by known plasmids bearing the LEU2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis (Van den Berg, 1990, Bio/Technology8:135). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed (Fleer et al., 1991, Bio/Technology 9:968-975).

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid molecule encoding an anti-ANGPT2 antibody or polypeptide chainthereof. Promoters suitable for use with prokaryotic hosts include phoApromoter, β-lactamase and lactose promoter systems, alkalinephosphatase, tryptophan (trp) promoter system, and hybrid promoters suchas the tac promoter. Other known bacterial promoters are also suitable.Promoters for use in bacterial systems also will contain a Shine-Dalgamo(S.D.) sequence operably linked to the DNA encoding the humanizedanti-ANGPT2 antibody.

Many eukaryotic promoter sequences are known. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Inducible promoters have the additional advantage of transcriptioncontrolled by growth conditions. These include yeast promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,derivative enzymes associated with nitrogen metabolism, metallothionein,glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 73,657. Yeastenhancers also are advantageously used with yeast promoters.

Anti-ANGPT2 antibody transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, or from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., 1982, Nature 297:598-601, disclosingexpression of human p-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

Another useful element that can be used in a recombinant expressionvector is an enhancer sequence, which is used to increase thetranscription of a DNA encoding an anti-ANGPT2 antibody by highereukaryotes. Many enhancer sequences are now known from mammalian genes(e.g., globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, an enhancer from a eukaryotic cell virus is used.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. See also Yaniv, 1982, Nature 297:17-18 for adescription of enhancing elements for activation of eukaryoticpromoters. The enhancer may be spliced into the vector at a position 5′or 3′ to the anti-ANGPT2 antibody-encoding sequence, but is preferablylocated at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) can also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-ANGPT2 antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein. In some embodiments, anti-ANGPT2 antibodies can beexpressed using the CHEF system. (See, e.g., U.S. Pat. No. 5,888,809;the disclosure of which is incorporated by reference herein.)

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-ANGPT2antibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-ANGPT2antibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells, including, e.g.,numerous baculoviral strains and variants and corresponding permissiveinsect host cells from hosts such as Spodoptera frugiperda(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),Drosophila melanogaster (fruitfly), and Bombyx mori (silk worm). Avariety of viral strains for transfection are publicly available, e.g.,the L-1 variant of Autographa californica NPV and the Bm-5 strain ofBombyx mori NPV, and such viruses may be used, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

The inventive anti-ANGPT2 antibodies or antigen-binding fragmentsthereof can also be incorporated in viral vectors, i.e. thepolynucleotide encoding for the anti-ANGPT2 antibody or antigen-bindingfragment thereof is introduced into the viral vector and then expressedin the body of the patient after infection with the virus.

In another aspect, expression of anti-ANGPT2 is carried out invertebrate cells. The propagation of vertebrate cells in culture (tissueculture) has become routine procedure and techniques are widelyavailable. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651), humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, (Graham et al., 1977, J. Gen Virol. 36: 59), babyhamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovarycells/-DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216; e.g., DG44), mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod.23:243-251), monkey kidney cells (CV1 ATCC CCL 70), African green monkeykidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells(HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo ratliver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals N.Y. Acad.Sci. 383: 44-68), MRC 5 cells, FS4 cells, and human hepatoma line (HepG2).

Host cells are transformed with the above-described expression orcloning vectors for anti-ANGPT2 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

The host cells used to produce anti-ANGPT2 antibody described herein maybe cultured in a variety of media. Commercially available media such asHam's F10 (Sigma-Aldrich Co., St. Louis, Mo.), Minimal Essential Medium((MEM), (Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), andDulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich Co.) aresuitable for culturing the host cells. In addition, any of the mediadescribed in one or more of Ham et al., 1979, Meth. Enz. 58: 44, Barneset al., 1980, Anal. Biochem. 102: 255, U.S. Pat. Nos. 4,767,704,4,657,866, 4,927,762, 4,560,655, 5,122,469, WO 90/103430, and WO87/00195 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as gentamicin), trace elements (defined as inorganiccompounds usually present at final concentrations in the micromolarrange), and glucose or an equivalent energy source. Other supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, the cells may bedisrupted to release protein as a first step. Particulate debris, eitherhost cells or lysed fragments, can be removed, for example, bycentrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology10:163-167 describes a procedure for isolating antibodies that aresecreted to the periplasmic space of E. coli. Briefly, cell paste isthawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants. A variety of methodscan be used to isolate the antibody from the host cell.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a typical purification technique. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human gammal, gamma2, orgamma4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth.62:1-13). Protein G is recommended for all mouse isotypes and for humangamma3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575). A matrix towhich an affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a C_(H3) domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, reverse phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, typically performed at low salt concentrations(e.g., from about 0-0.25M salt).

Also included are nucleic acids that hybridize under low, moderate, andhigh stringency conditions, as defined herein, to all or a portion(e.g., the portion encoding the variable region) of the nucleotidesequence represented by isolated polynucleotide sequence(s) that encodean ANGPT2-antibody or antibody fragment. The hybridizing portion of thehybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30 or50) nucleotides in length. The hybridizing portion of the hybridizingnucleic acid is at least 80%, e.g., at least 90%, at least 95%, or atleast 98%, identical to the sequence of a portion or all of a nucleicacid encoding an anti-ANGPT2 polypeptide (e.g., a heavy chain or lightchain variable region), or its complement. Hybridizing nucleic acids ofthe type described herein can be used, for example, as a cloning probe,a primer, e.g., a PCR primer, or a diagnostic probe.

In one embodiment, the present invention relates to an isolatedpolynucleotide or polynucleotides comprising:

a sequence encoding a heavy chain as shown in SEQ ID NO: 31 or a heavychain variable region as shown in SEQ ID NO: 3; and a sequence encodinga light chain as shown in SEQ ID NO. 32 or a light chain variable regionas shown in SEQ ID NO: 8,oran isolated polynucleotide or polynucleotides comprising a sequenceencoding a heavy chain as shown in SEQ ID NO: 33 or a heavy chainvariable region as shown in SEQ ID NO: 4; and a sequence encoding alight chain as shown in SEQ ID NO. 34 or a light chain variable regionas shown in SEQ ID NO: 9,oran isolated polynucleotide or polynucleotides comprising a sequenceencoding a heavy chain as shown in SEQ ID NO: 35 or a heavy chainvariable region as shown in SEQ ID NO: 5; and a sequence encoding alight chain as shown in SEQ ID NO. 36 or a light chain variable regionas shown in SEQ ID NO: 10,oran isolated polynucleotide or polynucleotides comprising a sequenceencoding a heavy chain as shown in SEQ ID NO: 37 or a heavy chainvariable region as shown in SEQ ID NO: 6; and a sequence encoding alight chain as shown in SEQ ID NO. 38 or a light chain variable regionas shown in SEQ ID NO: 11,oran isolated polynucleotide or polynucleotides comprising a sequenceencoding a heavy chain as shown in SEQ ID NO: 39 or a heavy chainvariable region as shown in SEQ ID NO: 7; and a sequence encoding alight chain as shown in SEQ ID NO. 40 or a light chain variable regionas shown in SEQ ID NO: 12.

It is to be understood that in said anti-ANGPT2 antibodies and antibodyfragments the nucleic acid sequence coding for the CDRs remain unchanged(unchanged with respect to the amino acid they encode, equivalents ofthe DNA sequence due to the degeneracy of codons are possible) but thesurrounding regions e.g. FR regions can be engineered.

Articles of Manufacture

In another aspect, an article of manufacture containing materials usefulfor the treatment of the disorders described above is included. Thearticle of manufacture comprises a container and a label. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers may be formed from a variety of materials such asglass or plastic. The container holds a composition that is effectivefor treating the condition and may have a sterile access port. Forexample, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle. The activeagent in the composition is the anti-ANGPT2 antibody or theantigen-binding fragment thereof. The label on or associated with thecontainer indicates that the composition is used for treating thecondition of choice. The article of manufacture may further comprise asecond container comprising a pharmaceutically-acceptable buffer, suchas phosphate-buffered saline, Ringer's solution, and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

The invention is further described in the following examples, which arenot intended to limit the scope of the invention.

EXAMPLES

Antibodies ANGPT2-opt-1, ANGPT2-opt-2, ANGPT2-opt-13, ANGPT2-opt-19, andANGPT2-opt-31 are characterized along with comparator antibodiesnesvacumab analog, MEDI3617 analog, and LC06. These comparatorantibodies were produced using standard procedures based on publishedsequences as described below.

TABLE 4 Comparator anti-ANGPT2 antibodies. Comparator Antibody Publishedsequence Nesvacumab analog (Regeneron) See the United States AdoptedName (USAN) file Heavy chain: SEQ ID NO. 53 number ZZ-34 (2012) fornesvacumab. For the Light chain: SEQ ID NO. 54 nesvacumab analogdescribed here, the lysine residue at position 219 in the heavy chain ofnesvacumab is replaced with arginine (K219R). MEDI3617 analog(Medimmune) The structure of the MEDI3617 analog used herein is Heavychain: SEQ ID NO. 55 based on the description in U.S. Pat. No.8,507,656. Light chain: SEQ ID NO. 56 LC06 (Roche) The structure of LC06used herein is based on the Heavy chain: SEQ ID NO. 57 description inU.S. Pat. No. 9,340,609. Light chain: SEQ ID NO. 58

Data for these antibodies are described below.

Example 1: Antibody Generation (Immunization)

Wild type CD1 mice are immunized with recombinant human and murineANGPT2 DNA, as well as human and murine ANGPT2 protein. CompleteFreund's Adjuvant, Freund's Incomplete Adjuvant, Titermax, or Gerbu areused as adjuvants at various points to augment antibody responses.Serology is then assessed by ELISA. Selected serologically positive miceare given a final boost before B-cell isolation. The mice selected allexhibit positive antibody titers in the sera. At the end of theimmunization regimen, splenocytes are harvested for recovery ofantigen-specific B-cells.

Example 2. Production of Humanized Antibodies

The chimeric lead CL-209881_VL was selected for further optimization.The chimeric lead has a variable light chain corresponding to SEQ ID NO:2 and a variable heavy chain corresponding to SEQ ID NO: 1. Thirty-threesequences (leads) were selected and optimized. Subsequent studies to theselection of six antibodies for further scale up.

Example 3. Sequence Liabilities in the CDRs

Sequences of the CDRs are checked for the presence for any potentialliabilities such as N-glycosylation sites, strong Deamidation motifs(NG, NS, NH, NA, ND, NT, NN), Aspartate isomerization motifs (DG),Fragmentation motifs (DG, DS), Cysteine. These amino acids or motifs canundergo chemical reaction and confer undesired heterogeneity to theproduct, also with the possibility of negatively impacting targetbinding and function. For these reasons, it is preferred to remove suchamino acids or motifs (if any) from the CDRs.

Example 4. Immunogenicity

Immunogenicity of sequences is evaluated in silico with an algorithmprovided through a license company EpiVax, Inc., Providence, R.I.EpiMatrix Treg-adj Score take into consideration T cell epitopes andTreg epitopes. The lower the immunogenicity score, the less likely asequence will be immunogenic. In general, a negative score is consideredlow risk of immunogenicity, while a positive score is viewed asindication for potential immunogenicity.

Example 5. Epitope Information Materials

Water (Sigma Aldrich, P/N 37877-4L)

Acetonitrile (Sigma Aldrich, P/N 34998-4L)

Formic acid (Fluka, P/N 94318)

Urea (Sigma Aldrich, P/N 51456-500G)

TCEP-HCl—10 g (Thermo Scientific—Pierce, P/N 20491)

Sodium Phosphate Disbasic (Sigma Aldrich, P/N S7907-100G)

Sodium Phosphate Monobasic (Sigma Aldrich, P/N S8282-500G)

ACQUITY UPLC BEH C18 VanGuard Pre-column, 130 Å, 1.7 μm, 2.1 mm X 5mm(Waters Technologies Corp, 186003975)

Poroszyme® Immobilized Pepsin Cartridge, 2.1 mm×30 mm(Life TechnologiesCorp, 2313100)

Acquity UPLC BEH C18 Column 1.7 um, 1 mm×50 mm (Waters, 186002344)

Solvent A: 0.1% Formic acid/99% water/1% acetonitrile

Solvent B: 0.1% Formic acid/5% water/95% acetonitrile

Water Buffer: H2O 10 mM sodium phosphate pH 7.4

Deuterium Buffer: D2O 10 mM sodium phosphate pH 7.4

Quench Buffer: Water 8 M Urea, 0.4M TCEP-HCl

In epitope mapping control samples, the antigen is run with and withoutantibody. To determine the list of antigen peptides, this protocol isfirst run using Water buffer in place of Deuterium buffer. 4 μL ofsample is mixed with 40 μL of Deuterium buffer. This mixture isincubated at 20° C. for multiple time points (1, 2, and 4 minutes). Then40 μL of the mixture is transferred to 40 μL of 4° C. quench buffer (4MUrea, 0.4M Tcep-HCl) and mixed. 60 μL of the quenched protein isinjected, where it's digested on the pepsin column for 2 minutes byflowing 200 μL/mL of solvent A: 0.1% Formic acid/99% water/1%acetonitrile. The subsequent peptides are desalted on the VanguardPre-column for 3 minutes. The peptic peptides are sent to a BEH C18reversed phase column inside the column/valve temperature controlledcompartment. A gradient solvent system consisting of solvent A: 0.1%Formic acid/99% water/1% acetonitrile and solvent B: 0.1% Formic acid/5%water/95% acetonitrile is utilized. The percentage of solvent B isincreased from 10% to 15% at 5.1 minute, to 50% at 11 minutes, to 90% at11.5 minutes held to 12.5 minutes, to 0% B at 13 minutes held to 14minutes. The chromatographic separation took place at 4° C. at a flowrate of 180 μl/min. After chromatographic separation, the sample entersthe Thermo Scientific Orbitrap Fusion mass spectrometer operated inpositive electrospray ionization mode. The employed method includesactivation types of CID and ETD when identifying control peptides,utilizing a resolution of 120,000, a minimum signal of 10,000, anisolation width of 1.0 and a normalized collision energy of 35.0 V. TheS-lens RF level is set at 60%. For control peptides, data collectiontype is profile for the full MS scan and centroid for the CID MS/MSdata. For Deuterated samples, no MS/MS is collected. Data is collectedover a mass range of 280-1800 Da. For raw LC-MS/MS fragmentation dataanalysis, control samples (with CID and ETD MS/MS) are analyzed usingProteome Discover 1.4 (Thermo Scientific) and PMi Byonic (ProteinMetrics) against the given sequence to generate a list of peptides andretention times. Raw data files are preprocessed and converted to ASCIIformat using proprietary in-house SHARC software. Identified peptidesare then matched and summarized using proprietary in-house SHAFTsoftware. Epitopes are determined by differences in average mass shiftinduced by binding after Deuterium labeling. On a peptide level,protection greater than 0.4 Da is considered significant.

Results of the epitope mapping are shown for an exemplary antibody ofthe invention (ANGPT2-opt-13) (FIG. 2), the nesvacumab analog, theMED3617 analog and LC06. Nesvacumab reportedly does not cross-react withANGPT1, whereas the MED3617 analog and LC06 reportedly cross-react withANGPT1. Specific binding sites of the antibodies to the FLD domain ofhuman ANGPT2 with SEQ ID NO: 50 (FIG. 2) are highlighted in dark grey.The data show that the comparator antibodies (nesvacumab analog,MED3617, and LC06) bind to epitopes that are different and distinct fromthe binding epitope of ANGPT2-opt-13.

Example 6. CDC Assay

The IgG heavy chain Fc region confers antibody effector functionsthrough interaction with C1q and therefore may have the ability toinduce complement-dependent cytotoxicity (CDC). This effect can beinvestigated in vitro by exposing target cells to complement in thepresence of an antibody that specifically binds to the target cell. TheCDC assay can be used to assess the activity of monoclonal antibodies tomediate CDC. (See “Mapping of the C1q binding site on Rituxan, achimeric antibody with a human IgG1 Fc,” Idusogie, Esohe E. et al.,Journal of Immunology (2000), 164 (8), 4178-4184; and “Utilization ofComplement-Dependent Cytotoxicity To Measure Low Levels of Antibodies:Application to Nonstructural Protein 1 in a Model of JapaneseEncephalitis Virus,” Konishi, Eiji et al., Clin Vaccine Immunol. (2008)Jan; 15(1): 88-94.)

CHO cells expressing membrane-bound human ANGPT2 (CHO-GPI-ANGPT2) arecultured in RPMI-1640 with the addition of HI FBS, 1× glutamax, and 1000μg/ml Geneticin (used as target cells). CDC activity is determined bymeasuring release of LDH from target cells using the CytotoxicityDetection Kit Plus from Roche®. Samples are set up in triplicate in96-well round bottom plates. Samples consist of 50 μl antibody, 50 μltarget cells (50,000/well), and 100 μl human complement (Cedarlane®), at1:12 final dilution in cytotoxicity media (Cedarlane®). Backgroundcontrol is 200 μl cytotoxicity media (Cedarlane®) only. Maximal releasecontrol (Tmax) is 50 μl target cells and 150 μl cytotoxicity media.Target cell control (Tspon) is 50 μl target cells and 150 μlcytotoxicity media. The plate is incubated at 37° C. in a humid CO₂incubator for 3 hours. Thirty minutes before the end of the incubation(after 2.5 hrs incubation), 10 μl Lysis Solution (provided in theCytotoxicity Detection Kit-Roche®) is added to the maximal release(Tmax) control wells. At the end of the incubation, 100 μl of thesupernatants are transferred into corresponding wells of a 96-wellflat-bottom plate for LDH detection. 100 μl of Reaction mixture(provided in the Cytotoxicity Detection Kit) is added to each well andthe plate is incubated at room temperature for 15 min in the dark. Atthe end of this second incubation, the reaction is stopped by adding 50μl of Stop solution (provided in the Cytotoxicity Detection Kit). Theabsorbance is measured at a wavelength of 490 nm with 650 nm asreference on Biotek® plate reader (Biotek, Synergy). CDC % is calculatedusing the equation:

% CDC=((Ab induced release)−[(T)_spon])/((T_max)−(T_spon))*100

The above-described CDC study is used to determine the cytotoxicitesresults of anti-ANG antibodies: ANGPT2-opt-13-IgG, nesvacumab analog,MEDI3617 analog, and LC06. The results of duplicate studies are shown inFIGS. 1A and 1B. The results show that the exemplary anti-ANGPT2antibody of the invention (ANGPT2-opt-13-IgG) is less cytoxic than eachof the nesvacumab analog, the MEDI3617 analog, and LC06.

Example 7. ANGPT2 Blocking Assay

Human ANGPT2 Dimer (CC-FLD) is pre-incubated with the testingantibodies. The ANGPT2/Antibody mixture is then incubated with HEK293human Tie2 cells at 4° C. After washing, the cells are stained withanti-His-AF647 (from GenScript) for detection of the His-tag on theANGPT2 protein. The binding of the secondarily labelled ANGPT2 to humanTie2 cells is detected by FLOW cytometry. The average of the duplicatevalues for each concentration point is used to derive the curve fitgraphs. Antibodies that prevent ANGPT2 from binding to the Tie2 cellsare considered blocking antibodies. The results are shown in FIGS. 3A-3Gand the EC50 values are shown in Table 5 based on the average ofduplicate values.

TABLE 5 ANGPT2 blocking assay. Anti-ANGPT2 EC50, antibody nM Ang2-opt-113.22 Ang2-opt-2 14.81 Ang2-opt-13 12.98 Ang2-opt-19 15.08 Ang2-opt-3113.03 Nevescumab analog 3.77

The results of the ANGPT2 blocking assay show antibodies of theinvention block ANGPT2 interaction with the receptor Tie2 on the cellsurface.

Example 8. Tie2 Phosphorylation Functional Assay

The Tie2 phosphorylation functional assay is carried out as describedbelow.

Unless otherwise indicated, the following reagents or materials areused:

HEK293/huTie2 cells;

0.25% trypsin/EDTA (Gibco cat.# 25200-056);

Poly-D-lysine coated black, clear bottom 96 well/plate (BioCoatcat#35460);

PathScan Sandwich ELISA lysis Buffer (Cell Signaling, cat#7018);

HALT protease and phosphatase inhibitor cocktail (Thermo Scientific,cat.#78443, lot# QK226996, 100× stock);

Activated sodium orthovanadate, 200 mM stock (Five photon, cat.#ActVO-4,lot# 26716-4);

Clear 96 well high bind polystyrene microplates (R&D Systems cat.#DY990, lot# 316940);

ELISA blocking buffer: Phosphate Buffered Saline (PBS) w/2% BSA, dilutedfrom 10% stock (R&D Systems, cat# DY995);

ELISA assay diluent: PBS w/1% BSA, diluted from 10% stock (R&D Systems,cat# DY995);

ELISA wash buffer concentrate: 25× stock (R&D Systems, cat.# WA126);

ELISA substrate reagent pack (R&D Systems, catDY999);

ELISA stop solution (R&D Systems, cat DY994); and

rhAngiopoietin-2 (R&D systems cat623-AN/CF; lot # SUL61 at 169 ug/mlstock).

Cell-Plating Medium

Dulbecco's Modified Eagle Medium (DMEM) with 4.5 g/l Glucose withL-Glutamine (500 ml) (Gibco cat.#11995-065);

Fetal Bovine Serum (FBS) (50 ml) (Hyclone catSH30071.03,Lot#AC10219630);

100 mM Non-Essential Amino Acids (NEAA) Solution (5ml) (Gibco:cat.#11140-050);

100 mM Sodium Pyruvate (5 ml) (Gibco cat#11360-070);

PenStrep (5 ml) (Gibco cat.#15140-122); and

1M N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (Hepes) (6.25 ml)(Gibco cat.#15630-080); Geneticin (10 ml) (Gibco cat#10131-035).

Starving Media

DMEM with 4,5 g/l Glucose with L-Glutamine;

FBS (25 ml) 5%; NEAA (5 ml);

Sodium Pyruvate (5 ml); PenStrep (2.5 ml);

Hepes (6.25 ml); and

Geneticin (10 ml) Dulbecco's Phosphate-Buffered Saline (dPBS), calciumand magnesium free (Gibco cat.#14190).

Cell Plating

HEK293/huTie2 cells are washed with PBS, detached with 0.25% Trypsin,and counted using a countess cell counter (Invitrogen). 5×10⁴cells areplated per well in a 96 well Ploy D-Lysine clear bottom tissue cultureplate in 100 ul Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS,NEAA, Sodium Pyruvate, PenStrep , Geneticin and HEPES. The cells areincubated overnight at 37° C., 5% CO₂ incubator. After about 18 hours,the cell-plating media is replaced with 100 ul of starving media(Dulbecco's Modified Eagle Medium (DMEM) with 5% FBS, NEAA, SodiumPyruvate, PenStrep, Geneticin and HEPES, returned the plate toincubator, and incubated overnight.

ELISA Plate Coating

The capture antibody (antiTie2 (AB33) from Cell signalling) is dilutedto a 1 ug/ml working solution in coating buffer (eBioscience). Theworking solution is immediately added to a 96 well high bindingpolystyrene microplate (R&D) to provide 100 ul per well. The wells areseal plated and incubated overnight at 4° C. The plate is washed 3 timeswith 300 ul per well by 1×ELISA wash buffer and then blocked with 200 ulblocking buffer for 2 hr at room temperature while shaking.

Cell Treatment

A stock solution of Ang2 (rhAngiopoietin-2 from R&D systems catalog#623-AN/CF; lot# SUL61) is diluted to 6 ug/ml in starving media. (Thestarving media is also used as antibody diluent.) Separately, solutionsof the anti-ANGPT2 antibodies are prepared by dilution to 66 nM followedby 1:3 serial dilution to 0.27 nM. Anti-ANGPT2 antibodies with rhAng2are incubated at room temperature for 30 min. Cell culture media (50 ul)is removed from each well in cell plate. The cells are treated with 50ul aliquots of the preincubated anti-ANGPT2 antibody solution for 20minutes at 37° C., 5% CO₂ incubator. The supernatant is discarded andthe cells are washed once with cold PBS (containing 1 mM activatedsodium orthovanadate). The wash buffer is discarded and PathScan lysisbuffer (cell signaling) with 1 mM activated sodium orthovanadate,protease and phosphatase inhibitors) is added to each cell. The plate isshaken at rapid speed for 1-3 hr at 4° C.

ELISA

The blocking buffer is removed from the ELISA plate, cell lysate isadded to each well, and the plate is incubated overnight at 4° C. withshaking. The plate is washed four times with ELISA wash buffer, and eachwell is treated with detection antibody: Biotin Conjugate-4G10 platinumanti-Phospotyrosine (from Millipore) at 1:300 dilution in ELISA assaydiluent. The plate is incubated for 2 hr on the shaker at roomtemperature and then washed four times with ELISA wash buffer.Streptavidin-HRP conjugated (from Millipore) diluted at 1:300 in ELISAassay diluent is added to each well and incubated on a shaker for 1 hrat room temperature. The plate is washed four times with ELISA washbuffer. Following the wash step, substrate solution from ELISA substratereagent pack (R&D Systems) is added to each well at room temperature for5-10 min. Stop solution is added followed by gentle tapping for 5minutes to ensure thorough mixing. The plate is then read with a platereader with absorbance at 450 nm corrected with 650 nm.

TABLE 6 Tie2 phosphorylation functional assay. Anti-ANGPT2 EC50, EC50,antibody nM nM Ang2-opt-1 3.0 3.2 Ang2-opt-2 1.8 1.9 Ang2-opt-13 4.2 4.0Ang2-opt-19 5.0 4.6 Ang2-opt-31 4.4 3.8 Ang2-opt-34 5.8 7.4 Nevescumabanalog 4.4 2.2 MEDI3617 analog — 2.7 LC06 1.3 —

The results of the Tie2 functional assay show that the anti-ANGPT2antibodies inhibit ANGPT2-induced Tie2 phosphorylation and can blockTie2-mediated phosphorylation and downstream signalling.

Example 9. Binding Affinity

The binding affinity to the various ANGPT2 analytes is determined bysurface plasmon resonance (SPR) using a ProteOn XPR36 (Bio-Rad). Unlessotherwise stated, all reagents are obtained from Bio-Rad. The runningbuffer for all assays and dilutions (except where stated) is phosphatebuffered saline (PBS)/ethylenediamine tetraacetic acid (EDTA) with 0.01%Tween20 (PBS-T-EDTA). PBS-T-EDTA is prepared by adding 100 μl of 100%Tween20 to 2 L of PBS-T-EDTA with an initial concentration of 0.005%Tween20 to make a final Tween20 concentration of 0.01%. The generallinearized model (GLM) sensorchip is normalized, pre-conditioned, andactivated with an equal mixture of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC)/N-hydroxysulfosuccinimide (s-NHS) in the horizontaldirection for 300 sec at a flow rate of 30 μl/min. Immobilization isthen is done with Recombinant Protein A/G (Thermo Scientific) (60 μg/mlin 10 mM acetate pH 4.5) in the horizontal direction for 300 sec at aflowrate of 30 μl/min resulting in about 4370-4875 response units (RUs)of Protein A/G on the surface. The sensorchip is then deactivated with1M ethanolamine HCl in the horizontal direction for 300 sec at aflowrate of 30 μl/min. The sensorchip is stabilized with 18 sec of 0.85%phosphoric acid at a flowrate of 100 μl/min 3 times horizontally and 3times vertically.

Each ANGPT2 antibody is captured on the Protein A/G surface verticallyfor 300 sec at a flowrate of 30 μl/min resulting in a capture level ofabout 2500 RU. The baseline is stabilized by injecting PBS-T-EDTA for 60sec at a flowrate of 100 μl/min horizontally with dissociation of 120sec. The analyte (e.g., HuANGPT2) is injected horizontally over thecaptured antibody for 300 sec at a flowrate of 30 μl/min and adissociation for 1800 sec. The concentrations of the analytes are 0 nM,6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM. The surface is regeneratedby injecting 0.85% phosphoric acid for 18 sec at a flowrate of 100μl/min one time horizontally and one time vertically. PBS-T-EDTA isinjected for 60 sec at a flowrate of 100 μl/min one time vertically andone time horizontally.

The interspot (interactions with sensor surface) and blank (PBS-T-EDTAwith 0.01% Tween20 or 0 nM of analyte (here HuANGPT2)) are subtractedfrom the raw data. Sensorgrams are then fit to 1:1 Langmuir binding toprovide on-rate (k_(a)), off-rate (k_(d)), and affinity (K_(D)) values.

The above procedure is used to measure the binding affinity to thefollowing analytes: human ANGPT1, human ANGPT2, cynomolgus (cyno)ANGPT2, rabbit ANGPT2, and rat ANGPT2. For the anti-ANGPT2 antibodies ofthe invention and the nesvacumab analog, no binding is observed in humanANGPT1 (K_(D)>100 nM). The results for binding to human ANGPT2 and cynoANGPT2 are shown in Table 7.

TABLE 7 Affinity binding properties of antibodies to ANGPT2 in human andcyno models. k_(a)(1/Ms) k_(d) (1/s) K_(D) (nM) k_(a)(1/Ms) k_(d) (1/s)kD (nM) Antibody human ANGPT2 cyno ANGPT2 CL_209881 1.51E+05 2.34E−050.155 1.64E+05 3.93E−05 0.240 ANGPT2-opt-1 1.44E+05 4.25E−05 0.2941.52E+05 5.48E−05 0.360 ANGPT2-opt-2 1.25E+05 8.11E−05 0.649 1.35E+058.45E−05 0.626 ANGPT2-opt-13 1.32E+05 1.89E−05 0.144 1.42E+05 2.08E−050.146 ANGPT2-opt-19 8.18E+04 5.83E−05 0.712 8.77E+04 7.40E−05 0.844ANGPT2-opt-31 1.25E+05 1.14E−05 0.091 1.35E+05 1.96E−05 0.145 Nesvacumab1.48E+05 1.93E−05 0.130 1.51E+05 1.93E−05 0.128 analog MEDI3617 6.14E+051.66E−02 27 NA NA NA analog LC06 1.05E+05 1.25E−02 12 NA NA NA NA = notavailable/not measured

For studies carried out in the rabbit ANGPT2 model, ANGPT2-opt-13 andnesvacumab analog exhibit K_(D) values of 0.133 nM and 0.137 nM,respectively.

For studies carried out in the rat model, the anti-ANGPT2 antibodies ofthe invention showed only weak binding (K_(D)≥118 nM), whereas thenesvacumab analog exhibits a K_(D) value of 0.159 nM.

The results show that the anti-ANGPT2 antibodies of the invention have ahigh affinity for human ANGPT2 and no measurable affinity for humanANGPT1.

The current disclosure contains, inter alia, the following items:

Embodiment 1: An anti-ANGPT2 antibody or an antigen-binding fragmentcomprising:

a heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 17 (H-CDR3), anda light chain variable region comprising the amino acid sequence of SEQID NO. 19 (L-CDR1); the amino acid sequence of SEQ ID NO. 22 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3),ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 14 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 17 (H-CDR3); anda light chain variable region comprising the amino acid sequence of SEQID NO. 19 (L-CDR1); the amino acid sequence of SEQ ID NO. 22 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3),ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 16 (H-CDR3); anda light chain variable region comprising the amino acid sequence of SEQID NO. 20 (L-CDR1); the amino acid sequence of SEQ ID NO. 23 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3),ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 16 (H-CDR3); anda light chain variable region comprising the amino acid sequence of SEQID NO. 21 (L-CDR1); the amino acid sequence of SEQ ID NO. 23 (L-CDR2);and the amino acid sequence of SEQ ID NO. 25 (L-CDR3),ora heavy chain variable region comprising the amino acid sequence of SEQID NO. 13 (H-CDR1); the amino acid sequence of SEQ ID NO. 15 (H-CDR2);and the amino acid sequence of SEQ ID NO. 16 (H-CDR3); anda light chain variable region comprising the amino acid sequence of SEQID NO. 20 (L-CDR1); the amino acid sequence of SEQ ID NO. 22 (L-CDR2);and the amino acid sequence of SEQ ID NO. 24 (L-CDR3).

Embodiment 2: The anti-ANGPT2 antibody of embodiment 1, wherein saidantibody comprises a variable heavy chain and a variable light chaincomprising the amino acid sequences of SEQ ID NO. 3 and SEQ ID NO. 8,respectively; SEQ ID NO. 4 and SEQ ID NO. 9, respectively; SEQ ID NO. 5and SEQ ID NO. 10, respectively; SEQ ID NO. 6 and SEQ ID NO. 11,respectively; or SEQ ID NO. 7 and SEQ ID NO. 12, respectively.

Embodiment 3: The anti-ANGPT2 antibody of embodiment 2, wherein saidantibody comprises a variable heavy chain and a variable light chainhaving at least 90%, at least 95%, at least 98%, or at least 99%identity to the amino acid sequences of SEQ ID NO. 3 and SEQ ID NO. 8,respectively; SEQ ID NO. 4 and SEQ ID NO. 9, respectively; SEQ ID NO. 5and SEQ ID NO. 10, respectively; SEQ ID NO. 6 and SEQ ID NO. 11,respectively; or SEQ ID NO. 7 and SEQ ID NO. 12, respectively.

Embodiment 4: The anti-ANGPT2 antibody of embodiment 1, wherein saidantibody comprises a heavy chain and a light chain comprising SEQ ID NO.31 and SEQ ID NO. 32, respectively; SEQ ID NO. 33 and SEQ ID NO. 34,respectively; SEQ ID NO. 35 and SEQ ID NO. 36; respectively; SEQ ID NO.37 and SEQ ID NO. 38; respectively; or SEQ ID NO. 39 and SEQ ID NO. 40.

Embodiment 5: The anti-ANGPT2 antibody of embodiment 4, wherein saidantibody comprises a heavy chain and a light chain having at least 90%,at least 95%, at least 98%, or at least 99% identity to the amino acidsequences of SEQ ID NO. 31 and SEQ ID NO. 32, respectively; SEQ ID NO.33 and SEQ ID NO. 34, respectively; SEQ ID NO. 35 and SEQ ID NO. 36;respectively; SEQ ID NO. 37 and SEQ ID NO. 38; respectively; or SEQ IDNO. 39 and SEQ ID NO. 40.

Embodiment 6: The anti-ANGTP2 antibody or antigen-binding fragmentthereof according to any of embodiments 1 to 5, wherein the anti-ANGTP2antibody or antigen-binding fragment thereof that binds to at least oneamino acid residue of SEQ ID NO: 51, or to SEQ ID NO: 51.

Embodiment 7: An anti-ANGTP2 antibody or antigen-binding fragmentthereof that binds to at least one amino acid residue within amino acidregions of the FLD domain of human ANGPT2 with the SEQ ID NO: 51, or toSEQ ID NO: 51.

Embodiment 8: An anti-ANGTP2 antibody or antigen-binding fragmentthereof that competes for binding to SEQ ID No: 51 with an anti-ANGTP2antibody according to any of embodiments 1 to 7.

Embodiment 9: An isolated polynucleotide having a sequence which encodesan antibody as defined in any of embodiments 1 to 8.

Embodiment10: A vector comprising the polynucleotide according toembodiment 9.

Embodiment 11: A host cell transformed or transfected with thepolynucleotide according to embodiment 9 or with the vector according toembodiment 10.

Embodiment 12:. A method for producing an antibody according to any ofembodiments 1 to 8, comprising (a) culturing a host cell according toembodiment 11 under conditions allowing the expression of the anbitodyaccording to any of embodiments 1 to 8 and (b) recovering the producedantibody from the culture.

Embodiment 13: A pharmaceutical composition comprising the anti-ANGPT2antibody or the antigen-binding fragment according to any of embodiments1 to 8, and a pharmaceutically acceptable carrier.

Embodiment 14: The pharmaceutical composition according to embodiment13, wherein the composition further comprises a second therapeutic agentselected.

Embodiment 15: The pharmaceutical composition according to embodiment 13or 14, wherein the composition is administered by a parenteral route,intravenous route or subcutaneous route of administration.

Embodiment 16: An anti-ANGPT2 antibody or the antigen-binding fragmentaccording to any of embodiments 1 to 8 for use as a medicament.

Embodiment 17: A method of treating diseases or disorders that can bealleviated by treatment with an anti-ANGPT2 antibody, the methodcomprising administering to a patient in need thereof a pharmaceuticallyeffective amount of the anti-ANGPT2 antibody or the antigen-bindingfragment according to any one of embodiments 1 to 8.

Embodiment 18: An anti-ANGPT2 antibody or the antigen-binding fragmentaccording to any of embodiments 1 to 8 for use in treating diseases ordisorders that can be alleviated by treatment with an anti-ANGPT2antibody.

Embodiment 19: Use of the anti-ANGPT2 antibody or the antigen-bindingfragment according to any of claims 1 to 8 in manufacture of amedicament for treating diseases or disorders that can be alleviated bytreatment with an anti-ANGPT2 antibody.

Embodiment 20: The method of embodiment 17, the anti-ANGPT2 antibody orthe antigen-binding fragment of embodiment 18, or the use of theanti-ANGPT2 antibody or the antigen-binding fragment of embodiment 19,wherein the disease or disorder is selected from the group consisting ofcardiac hypertrophy, myocardial infarction, ischemia, ischemicreperfusion injury, stroke hypertension, pulmonary arterialhypertension, idiopathic pulmonary arterial hypertension, trauma inducedbrain disorders, asthma, chronic obstructive pulmonary disease (COPD),rheumatoid arthritis, inflammatory bowel disease, multiplesclerosis,—preeclampsia and pregnancy-induced hypertension, sepsis,severe sepsis, septic shock, non-alcoholic steatohepatitis (NASH),cirrhosis, minimal change disease, focal segmental glomerulosclerosis(FSGS), nephrotic syndrome, diabetic kidney disease (DKD), chronickidney disease (CKD), diabetic renal insufficiency, end stage renaldisease, ischemia or an ischemic reperfusion injury, cancer,hepatocellular carcinoma, idiopathic pulmonary fibrosis (IPF),emphysema, acute lung injury (ALI), acute respiratory disease syndrome(ARDS), severe acute respiratory syndrome (SARS), Middle Easternrespiratory syndrome (MERS), vascular hyperpermeability (and associateddisorders), acute kidney injury, renal cell carcinoma, heart failure,lupus nephritis, Raynaud's, pancreatitis, peripheral artery disease,congenital heart disease, Dengue virus, malaria, hantavirus, edema,regeneration, lupus, interstitial lung disease, scleroderma,retinopathies, diabetic nephropathy, portal hypertension, varicesgrowth, and liver transplantation

Embodiment 21: The method of embodiment 20, the anti-ANGPT2 antibody orthe antigen-binding fragment of embodiment 20, or the use of theanti-ANGPT2 antibody or the antigen-binding fragment of embodiment 20,wherein the disease or disorder is selected from the group consisting ofchronic kidney disease, non-alcoholic steatohepatitis (NASH), andsepsis.

Embodiment 22: The method of embodiment 17 or 20, further comprisingadministering a second therapeutic agent selected.

Embodiment 23: The method of embodiment 17 or 20, wherein said antibodyis administered by a parenteral route, intravenous route or subcutaneousroute of administration.

Embodiment 24: A method of blocking the function of human ANGPT2 in ahuman patient, comprising administering to said human patient acomposition comprising the anti-ANGPT2 antibody or the antigen-bindingfragment according to any of embodiments 1 to 8 in an amount sufficientto block an ANGPT2 mediated response in said human patient.

Embodiment 25: An anti-ANGPT2 antibody or the antigen-binding fragmentaccording to any of embodiments 1 to 8 for use in blocking the functionof human ANGPT2.

Embodiment 26: Use of the anti-ANGPT2 antibody or the antigen-bindingfragment according to any of embodiments 1 to 8 in manufacture of amedicament for blocking the function of human ANGPT2.

Embodiment 27: An isolated polynucleotide encoding a heavy chainvariable region amino acid sequence or a light chain variable region,wherein the heavy chain variable region amino acid sequence comprisesany of SEQ ID NOs: 3 to 7, SEQ ID NOs: 13 to 17; SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39; a and the lightchain variable region amino acid sequence comprises any of SEQ ID NOs: 8to 12, SEQ ID NOs: 19 to 26, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 38, or SEQ ID NO: 40.

1-11. (canceled)
 12. An isolated polynucleotide encoding a heavy chainvariable region amino acid sequence or a light chain variable region,wherein the heavy chain variable region amino acid sequence comprisesany of SEQ ID NOs: 3 to 7, SEQ ID NOs: 13 to 17; SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39; a and the lightchain variable region amino acid sequence comprises any of SEQ ID NOs: 8to 12, SEQ ID NOs: 19 to 26, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 38, or SEQ ID NO: 40.